Enantiomers of selected fused heterocyclics and uses thereof

ABSTRACT

This invention relates to novel compounds having the structural formula (I) 
     
       
         
         
             
             
         
       
     
     and to their pharmaceutical compositions and to their methods of use. These novel compounds provide a treatment or prophylaxis of cancer.

This application is a continuation of U.S. application Ser. No.11/207,128, filed on 18 Aug. 2005, which claims the benefit of U.S.Application No. 60/602,366, filed on 18 Aug. 2004. The entire teachingsof U.S. application Ser. No. 11/207,128 and U.S. Application No.60/602,366 are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel fused heterocycles, theirpharmaceutical compositions and methods of use. In addition, the presentinvention relates to therapeutic methods for the treatment andprevention of cancers and to the use of these chemical compounds in themanufacture of a medicament for use in the treatment and prevention ofcancers.

BACKGROUND OF THE INVENTION

One sub-class of anti-cancer drugs (taxanes, vinca-alkaloids) now usedextensively in the clinic is directed at microtubules and block the celldivision cycle by interfering with normal assembly or disassembly of themitotic spindle (see Chabner, B. A., Ryan, D. P., Paz-Ares, l.,Garcia-Carbonero, R., and Calabresi, P: Antineoplastic agents. InHardman, J. G., Limbird, L. E., and Gilman, A. G., eds. Goodman andGilman's The Pharmacological Basis of Therapeutics, 10^(th) edition,2001, The MacGraw-Hill Companies, Inc). Taxol® (paclitaxel), one of themost effective drugs of this class, is a microtubule stabilizer. Itinterferes with the normal growth and shrinkage of microtubules thusblocking cells in the metaphase of mitosis. Mitotic block is oftenfollowed by slippage into the next cell cycle without having properlydivided, and eventually by apoptosis of these abnormal cells(Blagosklonny, M. V. and Fojo, T.: Molecular effects of paclitaxel:myths and reality (a critical review). Int J Cancer 1999, 83:151-156.).

Some of the side effects of treatment with paclitaxel are neutropeniaand peripheral neuropathy. Paclitaxel is known to cause abnormalbundling of microtubules in interphase cells. In addition, some tumortypes are refractory to treatment with paclitaxel, and other tumorsbecome insensitive during treatment. Paclitaxel is also a substrate forthe multi-drug resistance pump, P-glycoprotein ((see Chabner et al.,2001).

Thus, there is a need for effective anti-mitotic agents that have fewerside effects than anti-microtubule drugs, and also for agents that areeffective against taxane-resistant tumors.

Kinesins are a large family of molecular motor proteins, which use theenergy of adenosine 5′-triphosphate (ATP) hydrolysis to move in astepwise manner along microtubules. For a review, see Sablin, E. P.:Kinesins and microtubules: their structures and motor mechanisms. CurrOpin Cell Biol 2000, 12:35-41 and Schief, W. R. and Howard, J.:Conformational changes during kinesin motility. Curr Opin Cell Biol2001, 13:19-28.

Some members of this family transport molecular cargo along microtubulesto the sites in the cell where they are needed. For example, somekinesins bind to vesicles and transport them along microtubules inaxons. Several family members are mitotic kinesins, as they play rolesin the reorganization of microtubules that establishes a bipolar mitoticspindle. The minus ends of the microtubules originate at thecentrosomes, or spindle poles, whilst the plus ends bind to thekinetochore at the centromeric region of each chromosome. The mitoticspindle lines up the chromosomes at metaphase of mitosis and coordinatestheir movement apart and into individual daughter cells at anaphase andtelophase (cytokinesis). See Alberts, B., Bray, D., Lewis, J., Raff, M.,Roberts, K., and Watson, J. D., Molecular Biology of the Cell, 3^(rd)edition, Chapter 18, The Mechanics of Cell Division, 1994, GarlandPublishing, Inc. New York.

HsEg5 (homo sapiens Eg5) (Accession X85137; see Blangy, A., Lane H. A.,d'Heron, P., Harper, M., Kress, M. and Nigg, E. A.: Phosphorylation byp34cdc2 regulates spindle association of human Eg5, a kinesin-relatedmotor essential for bipolar spindle formation in vivo. Cell 1995, 83(7):1159-1169) or, KSP (kinesin spindle protein), is a mitotic kinesin whosehomologs in many organisms have been shown to be required for centrosomeseparation in the prophase of mitosis, and for the assembly of a bipolarmitotic spindle. For a review see Kashina, A. S., Rogers, G. C., andScholey, J. M.: The bimC family of kinesins: essential bipolar mitoticmotors driving centrosome separation. Biochem Biophys Acta 1997, 1357:257-271. Eg5 forms a tetrameric motor, and it is thought to cross-linkmicrotubules and participate in their bundling (Walczak, C. E., Vernos,I., Mitchison, T. J., Karsenti, E., and Heald, R.: A model for theproposed roles of different microtubule-based motor proteins inestablishing spindle bipolarity. Curr Biol 1998, 8:903-913). Severalreports have indicated that inhibition of Eg5 function leads tometaphase block in which cells display monastral spindles. Recently anEg5 inhibitor called monastrol was isolated in a cell-based screen formitotic blockers (Mayer, T. U., Kapoor, T. M., Haggarty, S. J., King, R.W., Schreiber, S. L., and Mitchison, T. J.: Small molecule inhibitor ofmitotic spindle bipolarity identified in a phenotype-based screen.Science 1999, 286: 971-974).

Monastrol treatment was shown to be specific for Eg5 over kinesin heavychain, another closely related motor with different functions (Mayer etal., 1999). Monastrol blocks the release of ADP (adenosine5′-diphosphate) from the Eg5 motor (Maliga, Z., Kapoor, T. M., andMitchison, T. J.: Evidence that monastrol is an allosteric inhibitor ofthe mitotic kinesin Eg5. Chem & Biol 2002, 9: 989-996 and DeBonis, S.,Simorre, J.-P., Crevel, I., Lebeau, L, Skoufias, D. A., Blangy, A.,Ebel, C., Gans, P., Cross, R., Hackney, D. D., Wade, R. H., andKozielski, F.: Interaction of the mitotic inhibitor monastrol with humankinesin Eg5. Biochemistry 2003, 42: 338-349) an important step in thecatalytic cycle of kinesin motor proteins (for review, see Sablin, 2000;Schief and Howard, 2001). Treatment with monastrol was shown to bereversible and to activate the mitotic spindle checkpoint which stopsthe progress of the cell division cycle until all the DNA is in placefor appropriate division to occur (Kapoor, T. M., Mayer, T. U.,Coughlin, M. L., and Mitchison, T. J.: Probing spindle assemblymechanisms with monastrol, a small molecule inhibitor of the mitotickinesin, Eg5. J Cell Biol 2000, 150(5): 975-988). Recent reports alsoindicate that inhibitors of Eg5 lead to apoptosis of treated cells andare effective against several tumor cell lines and tumor models (Mayeret al., 1999).

Although Eg5 is thought to be necessary for mitosis in all cells, onereport indicates that it is over-expressed in tumor cells (InternationalPatent Application WO 01/31335), suggesting that they may beparticularly sensitive to its inhibition. Eg5 is not present on themicrotubules of interphase cells, and is targeted to microtubules byphosphorylation at an early point in mitosis (Blangy et al., 1995). Seealso; Sawin, K. E. and Mitchison, T. J.: Mutations in the kinesin-likeprotein Eg5 disrupting localization to the mitotic spindle. Proc NatlAcad Sci USA 1995, 92(10): 4289-4293, thus monastrol has no detectableeffect on microtubule arrays in interphase cells (Mayer et al., 1999).Another report suggests that Eg5 is involved in neuronal development inthe mouse, but it disappears from neurons soon after birth, and thus Eg5inhibition may not produce the peripheral neuropathy associated withtreatment with paclitaxel and other anti-microtubule drugs (Ferhat, L.,Expression of the mitotic motor protein Eg5 in postmitotic neurons:implications for neuronal development. J Neurosci 1998, 18(19):7822-7835). Herein we describe the isolation of a class of specific andpotent inhibitors of Eg5, expected to be useful in the treatment ofneoplastic disease.

Certain pyrimidones have recently been described as being inhibitors ofKSP (WO 03/094839, WO 03/099211, WO 03/050122, WO 03/050064, WO03/049679, WO 03/049527, WO 04/078758, WO 04/106492 and WO 04/111058).

In accordance with the present invention, the present inventors havediscovered novel chemical compounds which possess Eg5 inhibitoryactivity and are accordingly useful for their anti-cell-proliferation(such as anti-cancer) activity and are therefore useful in methods oftreatment of the human or animal body.

SUMMARY OF THE INVENTION

An enantiomer of a compound of formula (I):

including a pharmaceutically acceptable salt or an in vivo hydrolysableester thereof,wherein:

X is selected from —C(CH₃)— or —S— provided that when X is —S— then Y is—C(CH₃)—;

Y is selected from —C(CH₃)— or —O— or —S— provided that when Y is—C(CH₃)— then X is not —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

R² and R³ are independently selected from H or C₁₋₃alkyl; wherein ifboth R² and R³ are selected from C₁₋₃alkyl they are identical;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl;

Z is optionally substituted phenyl, or optionally substitutedbenzothiophene wherein the number of optional substituents is 1 or 2 andeach is independently selected from F, Cl, Br, CH₃ or CH₂CH₃; and

“*I” represents a chiral center;

wherein said enantiomer is substantially free of the other enantiomer;and wherein the optical rotation of the enantiomer, when said enantiomeris dissolved at a concentration of 1 mg/ml in methanol, at 20.0° C.measured at 589 nM is (+).

The invention encompasses stereoisomers, enantiomers, invivo-hydrolysable precursors and pharmaceutically-acceptable salts ofcompounds of formula I, pharmaceutical compositions and formulationscontaining them, methods of using them to treat diseases and conditionseither alone or in combination with other therapeutically-activecompounds or substances, processes and intermediates used to preparethem, uses of them as medicaments, uses of them in the manufacture ofmedicaments and uses of them for diagnostic and analytic purposes.

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment, the present invention provides an enantiomer of anovel compound having structural formula (I):

including a pharmaceutically acceptable salt or an in vivo hydrolysableester thereof,wherein:

X is selected from —C(CH₃)— or —S— provided that when X is —S— then Y is—C(CH₃)—;

Y is selected from —C(CH₃)— or —O— or —S— provided that when Y is—C(CH₃)— then X is not —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

R² and R³ are independently selected from H or C₁₋₃alkyl; wherein ifboth R² and R³ are selected from C₁₋₃alkyl they are identical;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl;

Z is optionally substituted phenyl, or optionally substitutedbenzothiophene wherein the number of optional substituents is 1 or 2 andeach is independently selected from F, Cl, Br, CH₃ or CH₂CH₃; and

“*I” represents a chiral center;

wherein said enantiomer is substantially free of the other enantiomer;and wherein the optical rotation of the enantiomer, when said enantiomeris dissolved at a concentration of 1 mg/ml in methanol, at 20.0° C.measured at 589 nM is (+).

In a further aspect of the invention there is provided a compound offormula (I) having an optical rotation of (+):

including a pharmaceutically acceptable salt or an in vivo hydrolysableester thereof,wherein:

X is selected from C or S provided that when X is S then Y is C;

Y is selected from C or O or S provided that when Y is C then X is notC;

m is 0 or 1;

R¹ is F when m is 1;

R² and R³ are independently selected from H or C₁₋₃alkyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl;

Z is optionally substituted phenyl, or optionally substitutedbenzothiophene wherein the number of substituents is 1 or 2 and each isindependently selected from F, Cl, Br, CH₃ or CH₂CH₃.

In another embodiment, the present invention provides an (R) enantiomerof formula (Ia):

including a pharmaceutically acceptable salt or an in vivo hydrolysableester thereof,wherein:

X is selected from —C(CH₃)— or —S— provided that when X is —S— then Y is—C(CH₃)—;

Y is selected from —C(CH₃)— or —O— or —S— provided that when Y is—C(CH₃)— then X is not —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

R² and R³ are independently selected from H or C₁₋₃alkyl; wherein ifboth R² and R³ are selected from C₁₋₃alkyl they are identical;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl;

Z is optionally substituted phenyl, or optionally substitutedbenzothiophene wherein the number of optional substituents is 1 or 2 andeach is independently selected from F, Cl, Br, CH₃ or CH₂CH₃;

wherein said enantiomer is substantially free of the (S) enantiomer.

In another embodiment, the present invention provides an (S) enantiomerof formula (Ib):

including a pharmaceutically acceptable salt or an in vivo hydrolysableester thereof, wherein:

X is selected from —C(CH₃)— or —S— provided that when X is —S— then Y is—C(CH₃)—;

Y is selected from —C(CH₃)— or —O— or —S— provided that when Y is—C(CH₃)— then X is not —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

R² and R³ are independently selected from H or C₁₋₃alkyl; wherein ifboth R² and R³ are selected from C₁₋₃alkyl they are identical;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl;

Z is optionally substituted phenyl, or optionally substitutedbenzothiophene wherein the number of optional substituents is 1 or 2 andeach is independently selected from F, Cl, Br, CH₃ or CH₂CH₃.

wherein said enantiomer is substantially free of the (R) enantiomer.

In formula (I) the dotted line represents a single or a double bond —the bond between the nitrogen and whichever of X and Y is C is double,the other bond is a single bond.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein X is —C(CH₃)— or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein X is —S— or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Y is —C(CH₃)— or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Y is —S— or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Y is —O— or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Y is —S— and X is—C(CH₃)— or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Y is —O— and X is—C(CH₃)— or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Y is —C(CH₃)— and X is—S— or a pharmaceutically acceptable salt or an in vivo hydrolysableester thereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein m is 0 or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein m is 1 or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R² is H or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R² is methyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R² is ethyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R² is propyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R² is isopropyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R³ is methyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R³ is ethyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R³ is propyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R³ is isopropyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R² is H and R³ is methylor a pharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R² and R³ are methyl ora pharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein n is 2 or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein n is 3 or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R³ is H or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁴ is H or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁴ is methyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁴ is ethyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁴ is propyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁴ is isopropyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁵ is H or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁵ is methyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁵ is ethyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁵ is propyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁵ is isopropyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein R⁴ and R⁵ are both H orboth methyl, or R⁴ is H and R⁵ is isopropyl or a pharmaceuticallyacceptable salt or an in vivo hydrolysable ester thereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Z is optionallysubstituted phenyl or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Z is optionallysubstituted benzothiophene or a pharmaceutically acceptable salt or anin vivo hydrolysable ester thereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Z is 4-methylphenyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Z is benzothiophen-2-ylor a pharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Z is 4-chlorophenyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Z is 4-bromophenyl or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Z is4-methyl-3-fluorophenyl or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Z is 2,3-dichlorophenylor a pharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In an additional embodiment, the present invention provides anenantiomer of a compound of formula (I) wherein Z is 4-methylphenyl,benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl or a pharmaceuticallyacceptable salt or an in vivo hydrolysable ester thereof.

Particular values of variable groups are as follows. Such values may beused where appropriate with any of the definitions, claims orembodiments defined hereinbefore or hereinafter.

X is —C(CH₃)—.

X is S.

Y is C.

Y is S.

Y is O.

Y is —S— and X is —C(CH₃)—.

Y is —O— and X is —C(CH₃)—.

Y is —C(CH₃)— and X is —S—.

m is 0.

m is 1.

R² is H.

R² is methyl.

R² is ethyl.

R² is propyl.

R² is isopropyl.

R³ is methyl.

R³ is ethyl.

R³ is propyl.

R³ is isopropyl.

R² is H and R³ is methyl.

R² and R³ are methyl.

n is 2.

n is 3.

R³ is H.

R⁴ is H.

R⁴ methyl.

R⁴ is ethyl.

R⁴ is propyl.

R⁴ is isopropyl.

R⁵ is H.

R⁵ is methyl.

R⁵ is ethyl.

R⁵ is propyl.

R⁵ is isopropyl.

R⁴ and R⁵ are both H or both methyl, or R⁴ is H and R⁵ is isopropyl.

Z is optionally substituted phenyl.

Z is optionally substituted benzothiophene.

Z is 4-methylphenyl.

Z is benzothiophen-2-yl.

Z is 4-chlorophenyl.

Z is 4-bromophenyl.

Z is 4-methyl-3-fluorophenyl.

Z is 2,3-dichlorophenyl.

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl.

In a further aspect of the invention there is provided an enantiomer ofa compound of formula (I) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

X is selected from —C(CH₃)— or —S— provided that when X is —S— then Y is—C(CH₃)—;

Y is selected from —C(CH₃)— or —O— or —S— provided that when Y is—C(CH₃)— then X is not —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl;

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl; and

“*I” represents a chiral center;

wherein said enantiomer is substantially free of the other enantiomer;and wherein the optical rotation of the enantiomer, when said enantiomeris dissolved at a concentration of 1 mg/ml in methanol, at 20.0° C.measured at 589 nM is (+).

In a further aspect of the invention there is provided an (R) enantiomerof a compound of formula (Ia) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

X is selected from —C(CH₃)— or —S— provided that when X is —S— then Y is—C(CH₃)—;

Y is selected from —C(CH₃)— or —O— or —S— provided that when Y is—C(CH₃)— then X is not —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl; and

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl;

wherein said enantiomer is substantially free of the (S) enantiomer.

In a further aspect of the invention there is provided an (S) enantiomerof a compound of formula (Ib) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

X is selected from —C(CH₃)— or —S— provided that when X is —S— then Y is—C(CH₃)—;

Y is selected from —C(CH₃)— or —O— or —S— provided that when Y is—C(CH₃)— then X is not —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl; and

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl;

wherein said enantiomer is substantially free of the (R) enantiomer.

In a further aspect of the invention there is provided an enantiomer ofa compound of formula (I) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

Y is —S— and X is —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl;

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl; and

“*I” represents a chiral center;

wherein said enantiomer is substantially free of the other enantiomer;and wherein the optical rotation of the enantiomer, when said enantiomeris dissolved at a concentration of 1 mg/ml in methanol, at 20.0° C.measured at 589 nM is (+).

In a further aspect of the invention there is provided an (R) enantiomerof a compound of formula (Ia) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

Y is —S— and X is —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl; and

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl;

wherein said enantiomer is substantially free of the (S) enantiomer.

In a further aspect of the invention there is provided an (S) enantiomerof a compound of formula (Ib) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

Y is —S— and X is —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl; and

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl;

wherein said enantiomer is substantially free of the (R) enantiomer.

In a further aspect of the invention there is provided an enantiomer ofa compound of formula (I) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

Y is —O— and X is —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl;

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl; and

“*” represents a chiral center;

wherein said enantiomer is substantially free of the other enantiomer;and wherein the optical rotation of the enantiomer, when said enantiomeris dissolved at a concentration of 1 mg/ml in methanol, at 20.0° C.measured at 589 nM is (+).

In a further aspect of the invention there is provided an (R) enantiomerof a compound of formula (Ia) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

Y is —O— and X is —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl; and

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl;

wherein said enantiomer is substantially free of the (S) enantiomer.

In a further aspect of the invention there is provided an (S) enantiomerof a compound of formula (Ib) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

Y is —O— and X is —C(CH₃)—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl; and

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl;

wherein said enantiomer is substantially free of the (R) enantiomer.

In a further aspect of the invention there is provided an enantiomer ofa compound of formula (I) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

Y is —C(CH₃)— and X is —S—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl;

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl; and

“*I” represents a chiral center;

wherein said enantiomer is substantially free of the other enantiomer;and wherein the optical rotation of the enantiomer, when said enantiomeris dissolved at a concentration of 1 mg/ml in methanol, at 20.0° C.measured at 589 nM is (+).

In a further aspect of the invention there is provided an (R) enantiomerof a compound of formula (Ia) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

Y is —C(CH₃)— and X is —S—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl; and

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl;

wherein said enantiomer is substantially free of the (S) enantiomer.

In a further aspect of the invention there is provided an (S) enantiomerof a compound of formula (Ib) (as depicted above) including apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof,

wherein:

Y is —C(CH₃)— and X is —S—;

m is 0 or 1;

R¹ is F when m is 1;

one of R² and R³ is H and the other is methyl or both R² and R³ aremethyl;

n is 2 or 3;

R⁴ and R⁵ are independently selected from H or C₁₋₃alkyl; and

Z is 4-methylphenyl, benzothiophen-2-yl, 4-chlorophenyl, 4-bromophenyl,4-methyl-3-fluorophenyl or 2,3-dichlorophenyl;

wherein said enantiomer is substantially free of the (R) enantiomer.

In a further aspect of the invention there is provided a compound offormula (I) or a pharmaceutically acceptable salt thereof.

In an additional embodiment, the present invention provides a compoundof formula (I) or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof selected from:

-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-propyl}-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-propyl}-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-bromo-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-chloro-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-3-fluoro-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-2,3-dichloro-benzamide    hydrogen chloride;-   (+) Benzo[b]thiophene-2-carboxylic acid    (3-amino-propyl)-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]amide    hydrogen chloride;-   (+)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-isopropylamino-propyl)-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-benzamide    hydrogen chloride;-   (+)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-benzamide    hydrogen chloride;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-4-bromo-benzamide;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-3-fluoro-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide    hydrogen chloride;-   (+)    N-(3-Amino-propyl)-N-[1-(6-benzyl-3-methyl-7-oxo-6,7-dihydro-isothiazolo[4,5-d]pyrimidin-5-yl)-propyl]-4-methyl-benzamide.

In an additional embodiment, the present invention provides a compoundof formula (I) or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof selected from:

-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-propyl}-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-propyl}-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-bromo-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-chloro-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-3-fluoro-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-2,3-dichloro-benzamide;-   (+) Benzo[b]thiophene-2-carboxylic acid    (3-amino-propyl)-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]amide;-   (+)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-isopropylamino-propyl)-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;-   (+)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-benzamide;-   (+)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide;-   (+)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-benzamide;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-4-bromo-benzamide;-   (+)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-3-fluoro-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;-   (+)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;    or-   (+)    N-(3-Amino-propyl)-N-[1-(6-benzyl-3-methyl-7-oxo-6,7-dihydro-isothiazolo[4,5-d]pyrimidin-5-yl)-propyl]-4-methyl-benzamide.

In an additional embodiment, the present invention provides anenantiomer of formula (Ia) or a pharmaceutically acceptable salt or anin vivo hydrolysable ester thereof selected from:

-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-propyl}-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-propyl}-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-bromo-benzamide;-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-chloro-benzamide;-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-3-fluoro-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-2,3-dichloro-benzamide;-   (R) Benzo[b]thiophene-2-carboxylic acid    (3-amino-propyl)-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]amide;-   (R)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (R)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide;-   (R)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-isopropylamino-propyl)-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;-   (R)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-benzamide;-   (R)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide;-   (R)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-benzamide;-   (R)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide;-   (R)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-4-bromo-benzamide;-   (R)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-3-fluoro-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;-   (R)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;    or-   (R)    N-(3-Amino-propyl)-N-[1-(6-benzyl-3-methyl-7-oxo-6,7-dihydro-isothiazolo[4,5-d]pyrimidin-5-yl)-propyl]-4-methyl-benzamide.

In an additional embodiment, the present invention provides anenantiomer of formula (Ib) or a pharmaceutically acceptable salt or anin vivo hydrolysable ester thereof selected from:

-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-propyl}-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-propyl}-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-bromo-benzamide;-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-chloro-benzamide;-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-3-fluoro-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-2,3-dichloro-benzamide;-   (S) Benzo[b]thiophene-2-carboxylic acid    (3-amino-propyl)-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]amide;-   (S)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (S)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide;-   (S)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-isopropylamino-propyl)-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;-   (S)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-benzamide;-   (S)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide;-   (S)    N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-benzamide;-   (S)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide;-   (S)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-4-bromo-benzamide;-   (S)    N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino-propyl)-3-fluoro-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;-   (S)    N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide;    or-   (S)    N-(3-Amino-propyl)-N-[1-(6-benzyl-3-methyl-7-oxo-6,7-dihydro-isothiazolo[4,5-d]pyrimidin-5-yl)-propyl]-4-methyl-benzamide.

A particular embodiment of the invention refers to a compound of formula(I), (Ia) or (Ib) or a pharmaceutically acceptable salt thereof.

A compound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof, which is substantially free of itscorresponding (−) enantiomer.

The term “substantially free” refers to less than 10% of the otherisomer, more particulalry less than 5%, in particular less than 2%, moreparticularly less than 1%, particularly less then 0.5%, in particularless than 0.2%.

A compound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof having no more than about 1% w/w of thecorresponding (−) enantiomer.

A compound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof having no more than 1% w/w of thecorresponding (−) enantiomer.

A compound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof having no more than about 2% w/w of thecorresponding (−) enantiomer.

A compound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof having no more than 2% w/w of thecorresponding (−) enantiomer.

In an additional embodiment, the present invention provides a compoundof formula (I) or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof for use as a medicament.

Herein where the use of a compound of formula (I), or a method oftreatment comprising administering a compound of formula (I), or the useof a pharmaceutical composition comprising a compound of formula (I), isreferred to it is to be understood that “a compound of formula (I)”refers to (i) an enantiomer of a compound of formula (I); or (ii) an (R)enantiomer of formula (Ia); or (iii) an (S) enantiomer of formula (Ib).

According to a further aspect of the invention there is provided the useof a compound of the formula (I), or a pharmaceutically acceptable saltor an in vivo hydrolysable thereof, as defined hereinbefore in themanufacture of a medicament for use in the production of an Eg5inhibitory effect in a warm-blooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of the formula (I), or a pharmaceutically acceptable saltor an in vivo hydrolysable thereof, as defined hereinbefore in themanufacture of a medicament for use in the production of ananti-proliferative effect in a warm-blooded animal such as man.

According to this aspect of the invention there is provided the use of acompound of the formula (I), or a pharmaceutically acceptable salt or anin vivo hydrolysable thereof, as defined hereinbefore in the manufactureof a medicament for use in the production of an anti-cancer effect in awarm-blooded animal such as man.

According to a further feature of the invention, there is provided theuse of a compound of the formula (I), or a pharmaceutically acceptablesalt or an in vivo hydrolysable thereof, as defined herein before in themanufacture of a medicament for use in the treatment of carcinomas ofthe brain, breast, ovary, lung, colon and prostate, multiple myelomaleukemias, lymphomas, tumors of the central and peripheral nervoussystem, melanoma, fibrosarcoma, Ewing's sarcoma and osteosarcoma.

In an additional embodiment, the present invention provides the use of acompound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof, in the manufacture of a medicament forthe treatment or prophylaxis of disorders associated with cancer.

According to a further feature of this aspect of the invention there isprovided a method for producing an Eg5 inhibitory effect in awarm-blooded animal, such as man, in need of such treatment whichcomprises administering to said animal an effective amount of a compoundof formula (I), or a pharmaceutically acceptable salt or an in vivohydrolysable thereof, as defined above.

According to a further feature of this aspect of the invention there isprovided a method of producing an anti-proliferative effect in awarm-blooded animal, such as man, in need of such treatment whichcomprises administering to said animal an effective amount of a compoundof formula (I), or a pharmaceutically acceptable salt or an in vivohydrolysable thereof, as defined above.

According to a further feature of this aspect of the invention there isprovided a method for producing an anti-cancer effect in a warm-bloodedanimal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound offormula (I), or a pharmaceutically acceptable salt or an in vivohydrolysable thereof, as defined above.

In an additional embodiment, the present invention provides a method forthe prophylaxis treatment of cancers associated with comprisingadministering to a human in need of such treatment a therapeuticallyeffective amount of a compound of formula (I).

In a further embodiment the present invention provides a method for theprophylaxis treatment of cancers associated with comprisingadministering to a human in need of such treatment a therapeuticallyeffective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt or an in vivo hydrolysable thereof.

In an additional embodiment, the present invention provides a method ofproducing a cell cycle inhibitory (anti-cell-proliferation) effect in awarm-blooded animal, such as man, in need of such treatment withcomprises administering to said animal an effective amount of a compoundof formula (I).

In a further embodiment the present invention provides a method ofproducing a cell cycle inhibitory (anti-cell-proliferation) effect in awarm-blooded animal, such as man, in need of such treatment withcomprises administering to said animal an effective amount of a compoundof formula (I) or a pharmaceutically acceptable salt or an in vivohydrolysable thereof.

In an additional embodiment, the present invention provides a method forthe treatment of cancer comprising administering to a human atherapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof.

In a further embodiment the present invention provides a method for thetreatment of cancer comprising administering to a human atherapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt or an in vivo hydrolysable thereof.

In an additional embodiment, the present invention provides a method forthe treatment of breast cancer, colorectal cancer, ovarian cancer, lung(non small cell) cancer, malignant brain tumors, sarcomas, melanoma andlymphoma by administring a compound of formula (I) or a pharmaceuticallyacceptable salt or an in vivo hydrolysable ester thereof.

In a further embodiment the present invention provides a method for thetreatment of breast cancer, colorectal cancer, ovarian cancer, lung (nonsmall cell) cancer, malignant brain tumors, sarcomas, melanoma andlymphoma by administering a compound of formula (I) or apharmaceutically acceptable salt or an in vivo hydrolysable thereof.

According to an additional feature of this aspect of the invention thereis provided a method of treating carcinomas of the brain, breast, ovary,lung, colon and prostate, multiple myeloma leukemias, lymphomas, tumorsof the central and peripheral nervous system, melanoma, fibrosarcoma,Ewing's sarcoma and osteosarcoma, in a warm-blooded animal, such as man,in need of such treatment which comprises administering to said animalan effective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt or an in vivo hydrolysable thereof as defined hereinbefore.

In an additional embodiment, the present invention provides a method forthe treatment of cancer by administering to a human a compound offormula (I) or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof and an anti-tumor agent.

In an additional embodiment, the present invention provides apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof together with at least one pharmaceutically acceptable carrier,diluent or excipient.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises a compound of the formula (I), or apharmaceutically acceptable salt or an in vivo hydrolysable thereof, asdefined herein before in association with a pharmaceutically-acceptablediluent or carrier for use in the production of an Eg5 inhibitory effectin a warm-blooded animal such as man.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises a compound of the formula (I), or apharmaceutically acceptable salt or an in vivo hydrolysable thereof, asdefined herein before in association with a pharmaceutically-acceptablediluent or carrier for use in the production of an anti-proliferativeeffect in a warm-blooded animal such as man.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises a compound of the formula (I), or apharmaceutically acceptable salt or an in vivo hydrolysable thereof, asdefined herein before in association with a pharmaceutically-acceptablediluent or carrier for use in the production of an anti-cancer effect ina warm-blooded animal such as man.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises a compound of the formula (I), or apharmaceutically acceptable salt or an in vivo hydrolysable thereof, asdefined herein before in association with a pharmaceutically-acceptablediluent or carrier for use in the treatment of carcinomas of the brain,breast, ovary, lung, colon and prostate, multiple myeloma leukemias,lymphomas, tumors of the central and peripheral nervous system,melanoma, fibrosarcoma, Ewing's sarcoma and osteosarcoma in awarm-blooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of the formula (I), or a pharmaceutically acceptable saltor an in vivo hydrolysable thereof, as defined hereinbefore in theproduction of an Eg5 inhibitory effect in a warm-blooded animal such asman.

According to a further aspect of the invention there is provided the useof a compound of the formula (I), or a pharmaceutically acceptable saltor an in vivo hydrolysable thereof, as defined hereinbefore for use inthe production of an anti-proliferative effect in a warm-blooded animalsuch as man.

According to this aspect of the invention there is provided the use of acompound of the formula (I), or a pharmaceutically acceptable salt or anin vivo hydrolysable thereof, as defined hereinbefore for use in theproduction of an anti-cancer effect in a warm-blooded animal such asman.

According to a further feature of the invention, there is provided theuse of a compound of the formula (I), or a pharmaceutically acceptablesalt or an in vivo hydrolysable thereof, as defined herein before foruse in the treatment of carcinomas of the brain, breast, ovary, lung,colon and prostate, multiple myeloma leukemias, lymphomas, tumors of thecentral and peripheral nervous system, melanoma, fibrosarcoma, Ewing'ssarcoma and osteosarcoma.

In a further embodiment the present invention provides the use of acompound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable thereof, for the treatment or prophylaxis of disordersassociated with cancer.

The definitions set forth in this section are intended to clarify termsused throughout this application. The term “herein” means the entireapplication.

The term “C_(m-n)” or “C_(m-n) group” used alone or as a prefix, refersto any group having m to n carbon atoms.

The term “hydrocarbon” used alone or as a suffix or prefix, refers toany structure comprising only carbon and hydrogen atoms up to 14 carbonatoms.

The term “hydrocarbon radical” used alone or as a suffix or prefix,refers to any structure as a result of removing one or more hydrogensfrom a hydrocarbon.

The term “alkyl” used alone or as a suffix or prefix, refers tomonovalent straight or branched chain hydrocarbon radicals comprising,unless otherwise indicated, 1 to about 12 carbon atoms. Unless otherwisespecified, “alkyl” includes both saturated alkyl and unsaturated alkyl.Particularly “alkyl” refers to saturated alkyl. Particularly “C₁₋₃alkyl”refers to methyl, ethyl, propyl or isopropyl.

The term “five-membered” used as prefix refers to a group having a ringthat contains five ring atoms.

The term “substituted” used as a suffix of a first structure, moleculeor group, followed by one or more names of chemical groups refers to asecond structure, molecule or group, which is a result of replacing oneor more hydrogens of the first structure, molecule or group with the oneor more named chemical groups. For example, a “phenyl substituted bynitro” refers to nitrophenyl.

“RT” or “rt” means room temperature.

When any variable (e.g., R¹, R⁴ etc.) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-3 R¹, then saidgroup may optionally be substituted with 0, 1, 2 or 3 R¹ groups and R¹at each occurrence is selected independently from the definition of R¹.Also, combinations of substituents and/or variables are permissible onlyif such combinations result in stable compounds.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom on thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds.

As used herein, “pharmaceutically acceptable” is employed herein torefer to those compounds, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, phosphoric, and the like; and the saltsprepared from organic acids such as lactic, maleic, citric, benzoic,methanesulfonic, and the like. The pharmaceutically acceptable salts ofthe invention also include salts prepared with one of the followingacids benzene sulfonic acid, fumaric acid, methanesulfonic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid orL-tartaric acid.

Thus in one aspect of the invention there is provided a compound of theinvention, particularly one of the Examples described herein, as apharmaceutically acceptable salt, particularly a benzene sulfonic acid,fumaric acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid or L-tartaric acid salt.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred.

As used herein, “in vivo hydrolysable ester” means an in vivohydrolysable (or cleavable) ester of a compound of the formula (I) thatcontains a carboxy or a hydroxy group. For example amino acid esters,C₁₋₆alkoxymethyl esters like methoxymethyl; C₁₋₆alkanoyloxymethyl esterslike pivaloyloxymethyl; C₃₋₈cycloalkoxycarbonyloxy C₁₋₆yl esters like1-cyclohexylcarbonyloxyethyl, acetoxymethoxy, or phosphoramidic cyclicesters.

All chemical names were generated using a software system known asAutoNom Name accessed through ISIS draw.

Combinations

The anti-cancer treatment defined herein may be applied as a soletherapy or may involve, in addition to the compound of the invention,conventional surgery or radiotherapy or chemotherapy. Such chemotherapymay include one or more of the following categories of anti-tumouragents:

(i) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as alkylating agents (for examplecis-platin, carboplatin, oxaliplatin, cyclophosphamide, nitrogenmustard, melphalan, chlorambucil, busulphan, temozolomide andnitrosoureas); antimetabolites (for example gemcitabine and antifolatessuch as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,methotrexate, cytosine arabinoside and hydroxyurea); antitumourantibiotics (for example anthracyclines like adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere) polokinase inhibitors; andtopoisomerase inhibitors (for example epipodophyllotoxins like etoposideand teniposide, amsacrine, topotecan and camptothecin);(ii) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptordown regulators (for example fulvestrant), antiandrogens (for examplebicalutamide, flutamide, nilutamide and cyproterone acetate), LHRHantagonists or LHRH agonists (for example goserelin, leuprorelin andbuserelin), progestogens (for example megestrol acetate), aromataseinhibitors (for example as anastrozole, letrozole, vorazole andexemestane) and inhibitors of 5α-reductase such as finasteride;(iii) agents which inhibit cancer cell invasion (for examplemetalloproteinase inhibitors like marimastat and inhibitors of urokinaseplasminogen activator receptor function or inhibitors of SRC kinase(like4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyqyuinazoline(AZD0530; International Patent Application WO 01/94341) andN-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide(dasatinib, BMS-354825; J. Med. Chem., 2004, 47, 6658-6661)) orantibodies to Heparanase);(iv) inhibitors of growth factor function, for example such inhibitorsinclude growth factor antibodies, growth factor receptor antibodies (forexample the anti-erbb2 antibody trastuzumab [Herceptin™] and theanti-erbb1 antibody cetuximab [Erbitux, C225]), Ras/Raf signallinginhibitors such as farnesyl transferase inhibitors (for examplesorafenib (BAY 43-9006) and tipifarnib), tyrosine kinase inhibitors andserine/threonine kinase inhibitors, for example inhibitors of theepidermal growth factor family (for example EGFR family tyrosine kinaseinhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, AZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine(CI 1033) and erbB2 tyrosine kinase inhibitors such as lapatinib), forexample inhibitors of the platelet-derived growth factor family such asimatinib, and for example inhibitors of the hepatocyte growth factorfamily, c-kit inhibitors, abl kinase inhibitors, IGF receptor(insulin-like growth factor) kinase inhibitors and inhibitors of cellsignalling through MEK, AKT and/or PI3K kinases;(v) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, (for example the anti-vascularendothelial cell growth factor antibody bevacizumab [Avastin™], and VEGFreceptor tyrosine kinase inhibitors such as those disclosed inInternational Patent Applications WO 97/22596, WO 97/30035, WO 97/32856,WO 98/13354,4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814)) and compounds that workby other mechanisms (for example linomide, inhibitors of integrin αvβ3function and angiostatin), ang1 and 2 inhibitors;(vi) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO 00/40529,WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213, anti bcl2;(vii) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;(viii) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2,GDEPT (gene-directed enzyme pro-drug therapy) approaches such as thoseusing cytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi-drug resistance gene therapy;(ix) immunotherapy approaches, including for example ex-vivo and in-vivoapproaches to increase the immunogenicity of patient tumour cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte-macrophage colony stimulating factor, approaches to decreaseT-cell anergy, approaches using transfected immune cells such ascytokine-transfected dendritic cells, approaches usingcytokine-transfected tumour cell lines and approaches usinganti-idiotypic antibodies;x) cell cycle agents such as aurora kinase inhibitors (for examplePH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528, AX39459 and thespecific examples mentioned in WO02/00649, WO03/055491, WO2004/058752,WO2004/058781, WO2004/058782, WO2004/094410, WO2004/105764,WO2004/113324 which are incorporated herein by reference), and cyclindependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors (forexample the specific examples of WO01/14375, WO01/72717, WO02/04429,WO02/20512, WO02/66481, WO02/096887, WO03/076435, WO03/076436,WO03/076434, WO03/076433, WO04/101549 and WO04/101564 which areincorporated herein by reference); andxi) cytotoxic agents such as gemcitibine, topoisomerase 1 inhibitors(adriamycin, etoposide) and topoisomerase II inhibitors.

Such conjoint treatment may be achieved by way of the simultaneous,sequential or separate dosing of the individual components of thetreatment. Such combination products employ the compounds of thisinvention within the dosage range described hereinbefore and the otherpharmaceutically-active agent within its approved dosage range.

In a further aspect of the present invention there is provided acompound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof in combination with simultaneous,sequential or separate dosing of an anti-tumor agent or class selectedfrom the list herein above.

Therefore in a further embodiment the present invention provides amethod for the treatment of cancer by administering to a human acompound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof in combination with simultaneous,sequential or separate dosing of an anti-tumor agent or class selectedfrom the list herein above.

In a further aspect of the present invention there is provided the useof a compound of formula (I) or a pharmaceutically acceptable salt or anin vivo hydrolysable ester thereof in combination with simultaneous,sequential or separate dosing of an anti-tumor agent or class selectedfrom the list herein above for use in the manufacture of a medicamentfor use in the treatment of cancer.

In a further aspect of the present invention there is provided the useof a compound of formula (I) or a pharmaceutically acceptable salt or anin vivo hydrolysable ester thereof in combination with simultaneous,sequential or separate dosing of an anti-tumor agent or class selectedfrom the list herein above for use in the treatment of cancer.

The anti-cancer treatment defined herein may also include one or more ofthe following categories of pharmaceutical agents:

i) an agent useful in the treatment of anemia, for example, a continuouseythropoiesis receptor activator (such as epoetin alfa);ii) an agent useful in the treatment of neutropenia, for example, ahematopoietic growth factor which regulates the production and functionof neutrophils such as a human granulocyte colony stimulating factor,(G-CSF), for example filgrastim; andiii) an anti-emetic agent to treat nausea or emesis, including acute,delayed, late-phase, and anticipatory emesis, which may result from theuse of a compound of the present invention, alone or with radiationtherapy, suitable examples of such anti emetic agents includeneurokinin-1 receptor antagonists, 5H13 receptor antagonists, such asondansetron, granisetron, tropisetron, and zatisetron, GABAB receptoragonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid or Benecorten,an antidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol.

Such conjoint treatment may be achieved by way of the simultaneous,sequential or separate dosing of the individual components of thetreatment. Such conjoint treatment employs the compounds of thisinvention within the dosage range described hereinbefore and the otherpharmaceutically-active agent within its approved dosage range.

In a further aspect of the present invention there is provided acompound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof in combination with simultaneous,sequential or separate dosing of another pharmaceutical agent or classselected from the list herein above.

Therefore in a further embodiment the present invention provides amethod for the treatment of cancer by administering to a human acompound of formula (I) or a pharmaceutically acceptable salt or an invivo hydrolysable ester thereof in combination with simultaneous,sequential or separate dosing of another pharmaceutical agent or classselected from the list herein above.

In a further aspect of the present invention there is provided the useof a compound of formula (I) or a pharmaceutically acceptable salt or anin vivo hydrolysable ester thereof in combination with simultaneous,sequential or separate dosing of another pharmaceutical agent or classselected from the list herein above for use in the manufacture of amedicament for use in the treatment of cancer.

In a further aspect of the present invention there is provided the useof a compound of formula (I) or a pharmaceutically acceptable salt or anin vivo hydrolysable ester thereof in combination with simultaneous,sequential or separate dosing of another pharmaceutical agent or classselected from the list herein above for use in the treatment of cancer.

In addition to their use in therapeutic medicine, the compounds offormula (I) and their pharmaceutically acceptable salts are also usefulas pharmacological tools in the development and standardisation of invitro and in vivo test systems for the evaluation of the effects ofinhibitors of Eg5 in laboratory animals such as cats, dogs, rabbits,monkeys, rats and mice, as part of the search for new therapeuticagents.

In the above other pharmaceutical composition, process, method, use andmedicament manufacture features, the alternative and preferredembodiments of the compounds of the invention described herein alsoapply.

Formulations

Compounds of the present invention may be administered orally,parenteral, buccal, vaginal, rectal, inhalation, insufflation,sublingually, intramuscularly, subcutaneously, topically, intranasally,intraperitoneally, intrathoracially, intravenously, epidurally,intrathecally, intracerebroventricularly and by injection into thejoints.

The dosage will depend on the route of administration, the severity ofthe disease, age and weight of the patient and other factors normallyconsidered by the attending physician, when determining the individualregimen and dosage level as the most appropriate for a particularpatient.

An effective amount of a compound of the present invention for use intherapy of infection is an amount sufficient to symptomatically relievein a warm-blooded animal, particularly a human the symptoms ofinfection, to slow the progression of infection, or to reduce inpatients with symptoms of infection the risk of getting worse.

For preparing pharmaceutical compositions from the compounds of thisinvention, inert, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,dispersible granules, capsules, cachets, and suppositories.

A solid carrier can be one or more substances, which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, or tablet disintegrating agents; it can also be anencapsulating material.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

For preparing suppository compositions, a low-melting wax such as amixture of fatty acid glycerides and cocoa butter is first melted andthe active ingredient is dispersed therein by, for example, stirring.The molten homogeneous mixture is then poured into convenient sizedmolds and allowed to cool and solidify.

Suitable carriers include magnesium carbonate, magnesium stearate, talc,lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose,sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and thelike.

Some of the compounds of the present invention are capable of formingsalts with various inorganic and organic acids and bases and such saltsare also within the scope of this invention. Examples of such acidaddition salts include acetate, adipate, ascorbate, benzoate,benzenesulfonate, bicarbonate, bisulfate, butyrate, camphorate,camphorsulfonate, choline, citrate, cyclohexyl sulfamate,diethylenediamine, ethanesulfonate, fumarate, glutamate, glycolate,hemisulfate, 2-hydroxyethylsulfonate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, hydroxymaleate, lactate,malate, maleate, methanesulfonate, meglumine, 2-naphthalenesulfonate,nitrate, oxalate, pamoate, persulfate, phenylacetate, phosphate,diphosphate, picrate, pivalate, propionate, quinate, salicylate,stearate, succinate, sulfamate, sulfanilate, sulfate, tartrate, tosylate(p-toluenesulfonate), trifluoroacetate, and undecanoate. Base saltsinclude ammonium salts, alkali metal salts such as sodium, lithium andpotassium salts, alkaline earth metal salts such as aluminum, calciumand magnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, ornithine, and so forth. Also, basicnitrogen-containing groups may be quaternized with such agents as: loweralkyl halides, such as methyl, ethyl, propyl, and butyl halides; dialkylsulfates like dimethyl, diethyl, dibutyl; diamyl sulfates; long chainhalides such as decyl, lauryl, myristyl and stearyl halides; aralkylhalides like benzyl bromide and others. Non-toxicphysiologically-acceptable salts are preferred, although other salts arealso useful, such as in isolating or purifying the product.

The salts may be formed by conventional means, such as by reacting thefree base form of the product with one or more equivalents of theappropriate acid in a solvent or medium in which the salt is insoluble,or in a solvent such as water, which is removed in vacuo or by freezedrying or by exchanging the anions of an existing salt for another anionon a suitable ion-exchange resin.

In order to use a compound of the formula (I) or a pharmaceuticallyacceptable salt thereof for the therapeutic treatment (includingprophylactic treatment) of mammals including humans, it is normallyformulated in accordance with standard pharmaceutical practice as apharmaceutical composition.

In addition to the compounds of the present invention, thepharmaceutical composition of this invention may also contain, or beco-administered (simultaneously or sequentially) with, one or morepharmacological agents of value in treating one or more diseaseconditions referred to herein.

The term composition is intended to include the formulation of theactive component or a pharmaceutically acceptable salt with apharmaceutically acceptable carrier. For example this invention may beformulated by means known in the art into the form of, for example,tablets, capsules, aqueous or oily solutions, suspensions, emulsions,creams, ointments, gels, nasal sprays, suppositories, finely dividedpowders or aerosols or nebulisers for inhalation, and for parenteral use(including intravenous, intramuscular or infusion) sterile aqueous oroily solutions or suspensions or sterile emulsions.

Liquid form compositions include solutions, suspensions, and emulsions.Sterile water or water-propylene glycol solutions of the activecompounds may be mentioned as an example of liquid preparations suitablefor parenteral administration. Liquid compositions can also beformulated in solution in aqueous polyethylene glycol solution. Aqueoussolutions for oral administration can be prepared by dissolving theactive component in water and adding suitable colorants, flavoringagents, stabilizers, and thickening agents as desired. Aqueoussuspensions for oral use can be made by dispersing the finely dividedactive component in water together with a viscous material such asnatural synthetic gums, resins, methyl cellulose, sodium carboxymethylcellulose, and other suspending agents known to the pharmaceuticalformulation art.

The pharmaceutical compositions can be in unit dosage form. In suchform, the composition is divided into unit doses containing appropriatequantities of the active component. The unit dosage form can be apackaged preparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms.

Synthesis

The compounds of the present invention can be prepared in a number ofways well known to one skilled in the art of organic synthesis. Thecompounds of the present invention can be synthesized using the methodsdescribed below, together with synthetic methods known in the art ofsynthetic organic chemistry, or variations thereon as appreciated bythose skilled in the art. Such methods include, but are not limited to,those described below. All references cited herein are herebyincorporated in their entirety by reference.

The novel compounds of this invention may be prepared using thereactions and techniques described herein. The reactions are performedin solvents appropriate to the reagents and materials employed and aresuitable for the transformations being effected. Also, in thedescription of the synthetic methods described below, it is to beunderstood that all proposed reaction conditions, including choice ofsolvent, reaction atmosphere, reaction temperature, duration of theexperiment and workup procedures, are chosen to be the conditionsstandard for that reaction, which should be readily recognized by oneskilled in the art. It is understood by one skilled in the art oforganic synthesis that the functionality present on various portions ofthe molecule must be compatible with the reagents and reactionsproposed. Such restrictions to the substituents, which are compatiblewith the reaction conditions, will be readily apparent to one skilled inthe art and alternate methods must then be used.

The starting materials for the Examples contained herein are eithercommercially available or are readily prepared by standard methods fromknown materials. For example the following reactions are illustrationsbut not limitations of the preparation of some of the starting materialsand examples used herein.

All chiral purifications to separate the respective enantiomers werecarried out using a Chiralpak AD column (dimensions 250×20 mm, 10μcolumn) with a flow rate of 20 ml/min unless otherwise stated.Approximate elution times may vary depending on the concentration ofcompound loaded. Chiral purification generally resulted in 99% purity ofthe (+) enantiomer.

The signal refers to the direction of rotation of polarized light at 670nm as measured by an Advanced Laser Polarimeter (PDR-Chiral, Inc., LakePark, Fla.) at ambient temperature in the solvent composition indicated(reference Liu Y. S., Yu T., Armstrong D. W., LC-GC 17 (1999), 946-957).

EXAMPLES

The invention will now be illustrated by the following non limitingexamples in which, unless stated otherwise:

(i) temperatures are given in degrees Celsius (° C.); operations werecarried out at room or ambient temperature, that is, at a temperature inthe range of 18-30° C.;(ii) organic solutions were dried over anhydrous sodium sulphate;evaporation of solvent was carried out using a rotary evaporator underreduced pressure (600-4000 Pascals; 4.5-30 mmHg) with a bath temperatureof up to 60° C.;(iii) in general, the course of reactions was followed by TLC or MS andreaction times are given for illustration only;(iv) final products had satisfactory proton nuclear magnetic resonance(NMR) spectra and/or mass spectral data;(v) yields are given for illustration only and are not necessarily thosewhich can be obtained by diligent process development; preparations wererepeated if more material was required;(vii) when given, NMR data is in the form of delta values for majordiagnostic protons, given in parts per million (ppm) relative totetramethylsilane (TMS) as an internal standard, determined at 400 MHzusing deuterated chloroform (CDCl₃) as solvent unless otherwiseindicated;(vii) chemical symbols have their usual meanings; SI units and symbolsare used;(viii) solvent ratios are given in volume:volume (v/v) terms; and(ix) mass spectra were run with an electron energy of 70 electron voltsin the chemical ionization (CI) mode using a direct exposure probe;where indicated ionization was effected by electron impact (EI), fastatom bombardment (FAB); electrospray (ESP); or atmospheric pressurechemical ionization (APCI); values for m/z are given; generally, onlyions which indicate the parent mass are reported;(x) where a synthesis is described as being analogous to that describedin a previous example the amounts used are the millimolar ratioequivalents to those used in the previous example;(xi) the following abbreviations have been used:

THF tetrahydrofuran;

DMF N,N-dimethylformamide;

EtOAc ethyl acetate;

DCM dichloromethane; and

DMSO dimethylsulphoxide; and

(xii) a Vigreux column is a glass tube with a series of indentationssuch that alternate sets of indentations point downward at an angle of45 degree in order to promote the redistribution of liquid from thewalls to the center of the column; The Vigreux column used herein is 150mm long (between indents) with a 20 mm diameter and it was manufacturedby Lab Glass.

Method 1 2-(1-Ethoxy-ethylidene)-malononitrile

Triethyl orthoacetate (97 g, 0.6 mol), malononitrile (33 g, 0.5 mol) andglacial acetic acid (1.5 g) were placed in a 1 L flask equipped with astirrer, thermometer and a Vigreux column (20×1 in.) on top of which adistillation condenser was placed. The reaction mixture was heated andethyl alcohol began to distill when the temperature of the reactionmixture was about 85-90° C. After about 40 min., the temperature of thereaction mixture reached 140° C. Then the reaction was concentrated in arotary evaporator to remove the low-boiling materials and the residuewas crystallized from absolute alcohol to yield the pure product (62.2g, 91%) as a light yellow solid mp 91.6° C.

Method 2 (2E)-2-Cyano-3-ethoxybut-2-enethioamide

2-(1-Ethoxy-ethylidene)-malononitrile (method 1) (62 g, 0.45 mol) wasdissolved in anhydrous benzene (800 mL) and 1 mL of triethylamine wasadded as catalyst. The mixture was stirred and hydrogen sulfide wasbubbled into this solution for 40 min and a solid formed. Theprecipitated solid was filtered off and dried. The solid wasrecrystallized from absolute alcohol (100 mL) filtered and dried toisolate the pure (2E)-2-cyano-3-ethoxybut-2-enethioamide (19.3 g, 25%)as light brown crystals.

Method 3 (2E)-3-Amino-2-cyanobut-2-enethioamide

(2E)-2-Cyano-3-ethoxybut-2-enethioamide (method 2) (19.2 g, 0.136 mol)was dissolved in a saturated solution of ammonia in methanol (500 mL)and stirred at r.t. overnight. The reaction mixture was concentrated andthe residue was dissolved in hot water (600 mL) and the undissoved solidwas filtered and dried to recover 6 g of the starting thiocrotonamide.The aqueous solution on standing overnight provided the pure(2E)-3-amino-2-cyanobut-2-enethioamide (6.85 g, 63%) as off-whitecrystals. ¹H NMR (300 MHz, DMSO-d₆) δ 2.22 (s, 3H), 7.73 (bs, 1H), 8.53(bs, 1H), 9.01 (bs, 1H), 11.60 (bs, 1H).

Method 4 5-Amino-3-methylisothiazole-4-carbonitrile

To a stirred solution of (2E)-3-amino-2-cyanobut-2-enethioamide (method3) (6.83 g, 48.4 mmol) in methanol (300 mL) was added dropwise 13.6 mL(124 mmol.) of 30% hydrogen peroxide. The mixture was stirred at 60° C.for 4 h and evaporated to 60 mL in a rotary evaporator and cooled in anice-bath. The crystallized product was filtered off and recrystallizedfrom EtOAc to provide the pure product5-amino-3-methylisothiazole-4-carbonitrile (5.41 g, 80%) as a whitecrystalline solid. ¹H NMR (300 MHz, DMSO-d₆) δ 2.24 (s, 3H), 8.00 (bs,2H).

Method 5 N-(4-Cyano-3-methyl-isothiazol-5-yl)-butyramide

To a solution of 5-amino-3-methylisothiazole-4-carbonitrile (method 4)(5.31 g, 38.2 mmol) in DCM (200 mL) at 0° C., NEt₃ (5 g, 50 mmol) wasadded followed by the dropwise addition of a solution of the butyrylchloride (4.88 g, 45.8 mmol) in DCM (50 mL). After the completion of theaddition the reaction mixture was allowed to warm to r.t. and stirredovernight. The reaction mixture was washed with water (100 mL), 1N HCl(100 mL), brine (200 mL) and dried over Na₂SO₄. Concentration of the DCMlayer provided the crude product which was triturated from DCM/hexanes(1/10) and filtered off to isolate the pureN-(4-cyano-3-methyl-isothiazol-5-yl)-butyramide (7.57 g, 95%) as anorange solid.

Method 6 5-Butyrylamino-3-methyl-isothiazole-4-carboxylic Acid Amide

To a solution of N-(4-cyano-3-methyl-isothiazol-5-yl)-butyramide (method5) (4.18 g, 20 mmol) in 30% aqueous NH₄OH (250 mL), was added dropwise100 mL of hydrogen peroxide at r.t. After the completion of the additionthe reaction mixture was stirred at 60° C. overnight after which the TLCshowed the complete disappearance of SM. The reaction mixture was cooledand extracted with chloroform (3×100 mL). The organic layer was dried(Na₂SO₄) and concentrated to get the pure5-butyrylamino-3-methyl-isothiazole-4-carboxylic acid amide (2.9 g, 72%)as a white solid. ¹H NMR (300 MHz) δ 1.03 (t, 3H), 1.79 (m, 2H), 2.54(t, 3H), 2.69 (s, 3H), 5.97 (bs, 2H), 11.78 (bs, 1H).

Method 7 3-Methyl-6-propyl-5H-isothiazolo[5,4-d]pyrimidin-4-one

5-Butyrylamino-3-methyl-isothiazole-4-carboxylic acid amide (method 6)(1.9 g, 8.3 mmol) was suspended in 75 mL of 30% NH₃ and then was heatedto 140° C. for 4 h in a pressure reactor. The mixture was cooled andneutralized to pH 8. The precipitated3-methyl-6-propyl-5H-isothiazolo[5,4-d]pyrimidin-4-one was filtered off,washed with water (100 mL) and dried in vacuum oven at 40° C. overnightto get 800 mg (34%) of pure product. ¹H NMR (300 MHz) δ 1.03 (t, 3H),1.74 (m, 2H), 2.67 (t, 3H), 2.78 (s, 3H).

Method 8 5-Benzyl-3-methyl-6-propyl-5H-isothiazolo[5,4-d]pyrimidin-4-one

To a solution of 3-methyl-6-propyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 7) (800 mg, 3.8 mmol) in 20 mL of anhydrous DMF was added 1.38 g(10 mmol) of anhydrous K₂CO₃ followed by benzyl bromide (655 mg, 3.8mmol) and the mixture was stirred at room temperature overnight. The TLCof the reaction mixture showed the complete disappearance of the SM. Thereaction mixture was poured into ice cold water and extracted with EtOAc(3×100 mL). The combined extracts were washed with water (100 mL), brine(100 mL), dried (Na₂SO₄) and concentrated. The TLC and the ¹H NMR showedthe presence of two products N alkylated as well as O-alkylated productsin a ratio of 1:1. The products were separated by column (silica gel,116 g) chromatography using 10-20% EtOAc in hexanes. The desiredN-alkylated product5-benzyl-3-methyl-6-propyl-5H-isothiazolo[5,4-d]pyrimidin-4-one wasisolated as white crystalline solid (369 mg, 32%). ¹H NMR (300 MHz) δ0.96 (t, 3H), 1.71-1.84 (m, 2H), 2.73 (t, 3H), 2.81 (s, 3H), 5.38 (s,2H), 7.14-7.38 (m, 5H).

Methods 8a-8b

The following compounds were synthesized according to Method 8:

Method # Compound Name m/z Alkylating agent 8a5-(4-Fluoro-benzyl)-3-methyl- 318 (MH⁺) 4-fluorobenzyl6-propyl-5H-isothiazolo[5,4- bromide d]pyrimidin-4-one 8b5-(3-Fluoro-benzyl)-3-methyl- 318 (MH⁺) 3-fluorobenzyl6-propyl-5H-isothiazolo[5,4- bromide d]pyrimidin-4-one

Method 95-Benzyl-6-(1-bromo-propyl)-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one

To a solution of5-benzyl-3-methyl-6-propyl-5H-isothiazolo[5,4-d]pyrimidin-4-one (method8) (369 mg, 1.23 mmol) and sodium acetate (1 g) in acetic acid (5 mL) at100° C., a solution of the bromine (318 mg, 2 mmol) in acetic acid (10mL) was added dropwise over a period of 20 minutes. The reaction mixturewas cooled after the addition and the TLC (eluent 10% EtOAc in hexanes)and MS showed the complete disappearance of the SM and only the product.The reaction mixture was poured into ice water and extracted with EtOAc(3×60 mL) and the organic layers were combined and washed with 2% sodiumthiosulfate solution (60 mL), water (100 mL), brine (100 mL) and driedover Na₂SO₄. Concentration of the organic layer provided the pure5-benzyl-6-(1-bromo-propyl)-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one,(460 mg, 100%) as white crystalline solid. ¹H NMR (300 MHz) δ 0.76 (t,3H), 2.1-2.47 (m, 2H), 2.84 (s, 3H), 4.62 (t, 1H), 4.88 (d, 1H), 6.20(d, 1H), 7.10-7.40 (m, 5H).

Methods 9a-9b

The following compounds were synthesized according to Method 9:

Method # Compound Name m/z SM 9a 6-(1-bromopropyl)-5-[(4- 396, Method 8afluorophenyl)methyl]-3-methyl- 398 (MH⁺) isothiazolo[5,4-d]pyrimidin-4(5H)-one 9b 6-(1-bromopropyl)-5-[(3- 396, Method 8bfluorophenyl)methyl]-3-methyl- 398 (MH⁺) isothiazolo[5,4-d]pyrimidin-4(5H)-one

Method 10{3-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propylamino]-propyl}-carbamicAcid Tert-Butyl Ester

To a solution of5-benzyl-6-(1-bromo-propyl)-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 9) (0.46 g, 1.22 mmol) in anhydrous ethanol (20 mL), was addedtert-butyl 3-aminopropyl-carbamate (0.211 g, 1.22 mmol) followed by theaddition of anhydrous diisopropylethylamine (0.258 g, 2 mmol) and themixture was stirred at reflux for 16 hours. The TLC of the RM showed thecomplete disappearance of the starting bromide. The reaction mixture waspoured into ice water (200 mL) and extracted with EtOAc (3×100 mL). Theorganic layer was washed with water (100 mL), brine (100 mL) and dried(Na₂SO₄). Concentration of the organic layer provided the crude productwhich was purified by column (silica gel) chromatography using 30-50%EtOAc in hexanes to isolate the pure amine{3-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propylamino]-propyl}-carbamicacid tert-butyl ester (0.1 g, 17%) as a white foam. ¹H NMR (300 MHz) δ0.95 (t, 3H), 1.33 (t, 2H), 1.42 (s, 9H), 1.49-1.51 (m, 2H), 1.87-1.99(m, 1H), 2.35-2.45 (m, 1H), 2.83 (s, 3H), 2.92-3.20 (m, 2H), 3.64-3.70(m, 1H), 4.98 (d, 1H), 5.17 (bs, 1H), 5.85 (d, 1H), 7.10-7.40 (m, 5H).

Methods 10a-10d

The following compounds were synthesized according to Method 10:

Method # Compound Name m/z SM Amine 10a{3-({1-[5-(4-fluorobenzyl)-3-methyl-4- 490 (MH⁺) Method 9a tert-butyl 3-oxo-4,5-dihydro-isothiazolo[5,4- aminopropyl-d]pyrimidin-6-yl]-propyl}amino)- carbamate propyl}-carbamic acidtert-butyl ester 10b {3-({1-[5-(3-fluorobenzyl)-3-methyl-4- 490 (MH⁺)Method 9b tert-butyl 3- oxo-4,5-dihydro-isothiazolo[5,4- aminopropyl-d]pyrimidin-6-yl]-propyl}amino)- carbamate propyl}-carbamic acidtert-butyl ester 10c 5-Benzyl-6-[1-(3-dimethylamino- 400 (MH⁺) Method 9N,N- propylamino)-propyl]-3-methyl-5H- Dimethylpropane-isothiazolo[5,4-d]pyrimidin-4-one 1,3-diamine 10d{2-[1-(5-Benzyl-3-methyl-4-oxo-4,5- 458 (MH⁺) Method 9 (2-Amino-dihydro-isothiazolo[5,4-d]pyrimidin-6- ethyl)-carbamicyl)-propylamino]-ethyl}-carbamic acid acid tert-butyl tert-butyl esterester

Method 11{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicAcid Tert-Butyl Ester

To a solution of{3-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propylamino]-propyl}-carbamicacid tert-butyl ester (method 10) (0.1 g, 0.21 mmol) and triethylamine(0.303 g, 3 mmol) in DCM (20 mL) at r.t. was added dropwise a solutionof p-toluoyl chloride (0.1 g, 0.6 mmol) in DCM (10 mL). The resultingsolution was stirred at r.t. for 30 min. after which the TLC showed thedisappearance of the SM. The reaction mixture was diluted with DCM (60mL) washed with satd. NaHCO₃ (100 mL), water (100 mL), brine (100 mL)and dried (Na₂SO₄). Concentration of the organic layer provided thecrude product which was purified by column (silica gel) chromatographyusing 20-30% EtOAc in hexanes as eluent. Yield was 0.117 g (94%). m/z590 (MH⁺).

Methods 11a-11i

The following compounds were synthesized according to Method 11:

Method # Compound Name m/z SM Acylating agent 11a{3-[{1-[5-(4-Fluoro-benzyl)-3-methyl-4- 608 (MH⁺) Method4-methyl-benzoyl oxo-4,5-dihydro-isothiazolo[5,4- 10a chlorided]pyrimidin-6-yl]-propyl}-(4-methyl- benzoyl)-amino]-propyl}-carbamicacid tert-butyl ester 11b {3-[{1-[5-(3-Fluoro-benzyl)-3-methyl-4- 608(MH⁺) Method 4-methyl-benzoyl oxo-4,5-dihydro-isothiazolo[5,4- 10bchloride d]pyrimidin-6-yl]-propyl}-(4-methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester 11c{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5- 610 (MH⁺) Method 4-chloro-benzoyldihydro-isothiazolo[5,4-d]pyrimidin-6- 10 chlorideyl)-propyl]-(4-chloro-benzoyl)-amino]- propyl}-carbamic acid tert-butylester 11d {3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5- 608 (MH⁺) Method3-fluoro-4- dihydro-isothiazolo[5,4-d]pyrimidin-6- 10 methyl-benzoylyl)-propyl]-(3-fluoro-4-methyl-benzoyl)- chlorideamino]-propyl}-carbamic acid tert-butyl ester 11e{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5- 644, Method 2,3-dichloro-dihydro-isothiazolo[5,4-d]pyrimidin-6- 645, 10 benzoyl chlorideyl)-propyl]-(2,3-dichloro-benzoyl)- 646 (MH⁺) amino]-propyl}-carbamicacid tert-butyl ester 11f (3-{(Benzo[b]thiophene-2-carbonyl)-[1- 632(MH⁺) Method 1- (5-benzyl-3-methyl-4-oxo-4,5-dihydro- 10 benzothiophene-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]- 2-carbonylamino}-propyl)-carbamic acid tert-butyl chloride ester 11g{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5- 654, Method 4-bromo-benzoyldihydro-isothiazolo[5,4-d]pyrimidin-6- 656 (MH⁺) 10 chlorideyl)-propyl]-(4-bromo-benzoyl)-amino]- propyl}-carbamic acid tert-butylester 11h {2-[[1-(5-Benzyl-3-methyl-4-oxo-4,5- 576 (MH⁺) Method4-methyl-benzoyl dihydro-isothiazolo[5,4-d]pyrimidin-6- 10d chlorideyl)-propyl]-(4-methyl-benzoyl)-amino]- ethyl}-carbamic acid tert-butylester 11i N-[1-(5-Benzyl-3-methyl-4-oxo-4,5- 518 (MH⁺) Method4-methyl-benzoyl dihydro-isothiazolo[5,4-d]pyrimidin-6- 10c chlorideyl)-propyl]-N-(3-dimethylamino-propyl)- 4-methyl-benzamide

Method 12 Chiral Purification of (+)(3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicAcid Tert-Butyl Ester

100 mg of (+/−){3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 11) were dissolved in 2:1 IPA:hexanes andthe compound was purified using a Chiralpak AD, 250×20 mm, 10μ columnwith a flow rate of 20 ml/min with 80% hexane, 20% isopropanol (0.1%diethylamine) as eluent. Elution time:—10.42 min. Chiral purificationgenerally resulted in 99% purity of the (+) enantiomer.

Methods 12a-12i

The following compounds were chirally purified in same manner as (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 12):

Column Solvent (+) Enantiomer Method # Compound Name Type compositionretention time SM 12a (+) {3-[{1-[5-(4- Chiralpak 85% hexanes 10.7 min Method Fluoro-benzyl)-3- AD 15% isopropanol 11a methyl-4-oxo-4,5- 0.1%diethylamine dihydro- isothiazolo[5,4- d]pyrimidin-6-yl]-propyl}-(4-methyl- benzoyl)-amino]- propyl}-carbamic acid tert-butylester 12b (+) {3-[{1-[5-(3- Chiralpak 75% hexanes 7.6 min MethodFluoro-benzyl)-3- AD 25% isopropanol 11b methyl-4-oxo-4,5- 0.1%diethylamine dihydro- isothiazolo[5,4- d]pyrimidin-6-yl]-propyl}-(4-methyl- benzoyl)-amino]- propyl}-carbamic acid tert-butylester 12c (+) {3-[[1-(5-Benzyl- Chiralpak 80% hexanes 10.8 min  Method3-methyl-4-oxo-4,5- AD 20% isopropanol 11c dihydro- 0.1% diethylamineisothiazolo[5,4- d]pyrimidin-6-yl)- propyl]-(4-chloro- benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester 12d (+) {3-[[1-(5-Benzyl-Chiralpak 80% hexanes 8.6 min Method 3-methyl-4-oxo-4,5- AD 20%isopropanol 11d dihydro- 0.1% diethylamine isothiazolo[5,4-d]pyrimidin-6-yl)- propyl]-(3-fluoro-4- methyl-benzoyl)- amino]-propyl}-carbamic acid tert- butyl ester 12e (+) {3-[[1-(5-Benzyl- Chiralpak 90%hexanes 7.5 min Method 3-methyl-4-oxo-4,5- OD 5% methanol 11e dihydro-5% ethanol isothiazolo[5,4- 0.1% diethylamine d]pyrimidin-6-yl)-propyl]-(2,3-dichloro- benzoyl)-amino]- propyl}-carbamic acid tert-butylester 12f (+) (3- Chiralpak 50% hexanes 7.2 min Method{(Benzo[b]thiophene- AD 50% isopropanol 11f 2-carbonyl)-[1-(5- 0.1%diethylamine benzyl-3-methyl-4- oxo-4,5-dihydro- isothiazolo[5,4-d]pyrimidin-6-yl)- propyl]-amino}- propyl)-carbamic acid tert-butylester 12g (+) {3-[[1-(5-Benzyl- Chiralpak 75% hexanes 10.5 min  Method3-methyl-4-oxo-4,5- AD 25% isopropanol 11g dihydro- 0.1% diethylamineisothiazolo[5,4- d]pyrimidin-6-yl)- propyl]-(4-bromo- benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester 12h (+) {2-[[1-(5-Benzyl-Chiralpak 80% hexanes 11.8 min  Method 3-methyl-4-oxo-4,5- AD 20%isopropanol 11h dihydro- 0.1% diethylamine isothiazolo[5,4-d]pyrimidin-6-yl)- propyl]-(4-methyl- benzoyl)-amino]- ethyl}-carbamicacid tert-butyl ester 12i (+) N-[1-(5-Benzyl-3- Chiralpak 90% hexanes9.5 min Method methyl-4-oxo-4,5- AD 10% isopropanol 11i dihydro- 0.1%diethylamine isothiazolo[5,4- d]pyrimidin-6-yl)- propyl]-N-(3-dimethylamino- propyl)-4-methyl- benzamide Example A-10 Chiralpurification generally resulted in 99% purity of the (+) enantiomer.

Method 13 and Example A-1 (+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamideHydrogen Chloride

(+){3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicacid tert-butyl ester (method 12) (0.117 g, 0.19 mmol) was dissolved in2M HCl in ether and the mixture was stirred at r.t. for 20 h. Theprecipitated product was filtered off and washed with ether and dried invacuo to yield the pure (+)N-(3-amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamidechloride salt (91 mg, 87%). White powder, mp. 127.8-129.2° C. m/z 490(MH⁺), ¹H NMR (DMSO-d₆, 500 MHz, 96° C.) δ: 0.63 (t, 3H), 1.40-1.74 (m,2H), 1.75-1.96 (m, 1H), 2.05-2.20 (m, 1H), 2.39 (s, 3H), 2.46 (t, 2H),2.72 (s, 3H), 3.36 (t, 2H), 4.83 (d, 1H), 5.50 (bs, 1H), 5.77 (d, 1H),6.95-7.37 (m, 9H), 7.79 (bs, 3H).

Methods 13a-13h

The following compounds were synthesized according to Method 13:

Method # Compound Name m/z SM 13a(+)N-(3-Amino-propyl)-N-[1-(5-{4-fluorobenzyl}-3- 508 (MH⁺) Methodmethyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)- 12apropyl]-4-methyl-benzamide hydrogen chloride Example A-2 13b(+)N-(3-Amino-propyl)-N-[1-(5-{3-fluorobenzyl}-3- 508 (MH⁺) Methodmethyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)- 12bpropyl]-4-methyl-benzamide hydrogen chloride Example A-3 13c(+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo- 510 (MH⁺) Method4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4- 12cchloro-benzamide hydrogen chloride Example A-5 13d(+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo- 508 (MH⁺) Method4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-3- 12dfluoro-4-methyl-benzamide hydrogen chloride Example A-6 13e(+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo- 544, Method4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-2,3- 545, 12edichloro-benzamide hydrogen chloride 546 (MH⁺) Example A-7 13f(+)Benzo[b]thiophene-2-carboxylic acid (3-amino-propyl)- 532 (MH⁺)Method [1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4- 12fd]pyrimidin-6-yl)-propyl]amide hydrogen chloride Example A-8 13g(+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo- 554, Method4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4- 556 (MH⁺) 12gbromo-benzamide hydrogen chloride Example A-4 13h(+)N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5- 476 (MH⁺) Methoddihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl- 12hbenzamide hydrogen chloride Example A-9 13gN-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5- 490 (MH⁺) Methoddihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl- 11 benzamidehydrogen chloride

Method 14N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-isopropylamino-propyl)-4-methyl-benzamide

To a solution ofN-(3-amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-4-methyl-benzamidehydrogen chloride (method 13g) (1.24 g, 2.54 mmol), in the presence ofmolecular sieves (2 g,) was added acetone (1 mL) and the mixture wasstirred at room temperature for 2 h. Analysis of the reaction mixture byMS showed the completion of the schiff's base formation. To this mixturewas added two drops of acetic acid followed by sodiumtriacetoxyborohydride (220 mg) and the mixture was stirred overnight.The reaction mixture was filtered and the filtrate was washed withwater, dried (Na₂SO₄) and concentrated to get the crude product whichwas purified by column chromatography (silica gel) using 0-30% EtOAc inhexanes.N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-isopropylamino-propyl)-4-methyl-benzamidewas isolated as a white foam. Yield 0.206 g (15%). m/z 532 (MH+); ¹H NMR(DMSO-d₆, 96° C.) δ: 0.65 (t, 3H), 1.05 (d, 6H), 1.26-1.48 (m, 1H),1.65-1.70 (m, 1H), 1.80-1.98 (m, 1H), 2.00-2.17 (m, 1H), 2.35 (s, 3H),2.63 (b, 2H), 2.80 (s, 3H), 3.05 (b, 1H), 3.40 (t, 2H), 4.90 (d, 1H),5.50 (bs, 1H), 5.80 (d, 1H), 7.35-7.00 (m, 9H).

Method 15 and Example B-1 Chiral purification of (+)N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-N-(3-isopropylamino-propyl)-4-methyl-benzamide

The following compound was chirally purified in same manner as (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 12). Chiral purification generallyresulted in 99% purity of the (+) enantiomer.

Column Solvent (+) Enantiomer Method # Compound Name Type compositionretention time SM 15 (+) N-[1-(5-Benzyl-3- Chiralpak 85% hexanes 8.0 minMethod methyl-4-oxo-4,5- AD 15% 14 dihydro- isopropanol isothiazolo[5,4-0.1% d]pyrimidin-6-yl)- diethylamine propyl]-N-(3- isopropylamino-propyl)-4-methyl- benzamide Example B-1

Method 16 5-Butyrylamino-3-methyl-isoxazole-4-carboxylic Acid Amide

A mixture of 5-amino-3-methyl-isoxazole-4-carboxylic acid amide (2 g,14.18 mmol) in 10 ml of butyric anhydride was stirred at 150° C. for0.5˜1 h. The brown solution was diluted with hexane (100 ml) and cooledto room temperature. The solid crushed out from the mixture was filteredand washed with hexane, dried in vacuo. The title amide (2.6 g) wasobtained as white solid.

Method 17 3-Methyl-6-propyl-5H-isoxazolo[5,4-d]pyrimidin-4-one

A suspension of 5-butyrylamino-3-methyl-isoxazole-4-carboxylic acidamide (method 16) (2.6 g, split into 20 vials) in 3.5 ml of 2N NaOH aqwas subjected to microwave irradiation under the temperature of 140° C.for 20 min. The resulting solution was cooled with an ice bath, and thepH was adjusted to 1˜3 with concentrated HCl. The crushed out solid wasfiltered, washed with water, dried over vacuum at 40° C. overnight. Thetitle pyrimidinone (1.749 g) was obtained as white solid. ¹H NMR(DMSO-d₆): 0.91 (t, 3H), 1.71 (m, 2H), 2.44 (s, 3H), 2.64 (t, 2H), 12.78(s, 1H).

Method 18 5-Benzyl-3-methyl-6-propyl-5H-isoxazolo[5,4-d]pyrimidin-4-one

A suspension of 3-methyl-6-propyl-5H-isoxazolo[5,4-d]pyrimidin-4-one(method 17) (1.698 g, 8.8 mmol), benzylbromide (1.5 g, 8.8 mmol),potassium carbonate (2.43 g, 17.6 mmol) in 10 ml DMF was stirred at roomtemperature overnight. The mixture was diluted with water, extractedwith EtOAc (50 ml×3), the combined organic phases were dried,concentrated, purified by flash column chromatography (elute:hexane-EtOAc=5:1). 1.69 g (68%) of the title compound was obtained aswhite solid. ¹H NMR (DMSO-d₆): 0.80 (t, 3H), 1.61 (m, 2H), 2.43 (s, 3H),2.73 (t, 2H), 5.35 (s, 2H), 7.12-7.35 (m, 5H).

Method 195-Benzyl-6-(1-bromo-propyl)-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one

A solution of5-benzyl-3-methyl-6-propyl-5H-isoxazolo[5,4-d]pyrimidin-4-one (method18) (3.167 g, 11.2 mmol) and sodium acetate (4.59 g, 56 mmol, 5 eq) inglacial acetic acid (26 ml) was treated with a preformed brominesolution (0.7 ml bromine in 10 ml of glacial acetic acid) (8.64 ml, 22.4mmol, 2 eq). The mixture was stirred at 100° C. for 24 hrs. Excessbromine (8.64 ml, 22.4 mmol, 2 eq) was added to the mixture. The mixturewas then stirred at 100° C. for another 24 hrs. Water was added to thereaction mixture, followed by aq. potassium carbonate. The mixture wasextracted with DCM (50 ml×3), the combined organic phases were washedwith water and dried, then concentrated to give the crude product whichwas purified by flash chromatography (elute: hexane-EtOAc). 2.5 gproduct was furnished as a white solid. ¹H NMR (DMSO-d₆): 0.79 (t, 3H),2.18 (m, 1H), 2.35 (m, 1H), 2.58 (s, 3H), 5.12 (t, 1H), 5.25 (d, 1H),5.80 (d, 1H), 7.27-7.42 (m, 5H).

Method 20{3-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propylamino]-propyl}-carbamicAcid Tert-Butyl Ester

To a suspension of5-benzyl-6-(1-bromo-propyl)-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one(method 19) (2.8 g, 7.73 mmol) and potassium carbonate (2.67 g, 19.38mmol) in acetonitrile (100 ml) was addedtert-butyl-N-(3-aminopropyl)-carbamate (1.345 g, 7.73 mmol). The mixturewas stirred at 100° C. overnight. Water (30 ml) was added to themixture, which was extracted with EtOAc (3×50 ml). The combined organicphases were washed with brine (10 ml), dried, concentrated to obtain thecrude title amine which was purified by flash chromatography column(elute: EtOAc-hexane=1-4˜1-1) to give 2.6 g (74%) of product as whitesolid. ¹H NMR (DMSO-d₆): 0.85 (t, 3H), 1.32 (m, 2H), 1.41 (s, 9H), 1.58(m, 1H), 1.65 (m, 1H), 2.09 (m, 1H), 2.40 (m, 1H), 2.60 (s, 3H), 2.81(m, 2H), 3.29 (m, 1H), 3.75 (m, 1H), 5.42 (d, 1H), 5.63 (d, 1H), 6.72(br, 1H), 7.25-7.45 (m, 5H).

Method 21(3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicAcid Tert-Butyl Ester

A solution of{3-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propylamino]-propyl}-carbamicacid tert-butyl ester (method 20) (135 mg, 0.297 mmol) in DCM (4 ml) wasadded to 4-methyl-benzoyl chloride (46 mg, 0.297 mmol) followed bytriethylamine (60 mg, 0.594 mmol). The mixture was stirred at roomtemperature for 1 hr. Then diluted with DCM, washed with saturated aq.sodium bicarbonate. The organic phase was dried, filtered, andconcentrated. The crude oil was purified by flash column chromatography(solvent: EtOAc-hexane) to furnish(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (130 mg) (76% yield) as a white solid. ¹H NMR (500MHz, 100° C., DMSO-d₆): 0.71 (t, 3H), 1.12 (m, 1H), 1.35 (s, 9H), 1.47(m, 1H), 1.92 (m, 1H), 2.14 (m, 1H), 2.37 (s, 3H), 2.56 (s, 3H), 2.57(m, 2H), 3.29 (m, 2H), 5.01 (d, 1H), 5.68 (m, br, 1H), 5.79 (d, 1H),6.06 (br, 1H), 7.14-7.36 (m, 9H).

Method 22 Chiral Purification of (+)(3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicAcid Tert-Butyl Ester

The following compound was chirally purified in same manner as (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 12). Chiral purification generallyresulted in 99% purity of the (+) enantiomer.

Column Solvent (+) Enantiomer Method # Compound Name Type compositionretention time SM 22 (+) (3-[[1-(5-Benzyl-3- Chiralpak 70% hexanes 12.1min Method methyl-4-oxo-4,5- AD 30% 21 dihydro-isoxazolo[5,4-isopropanol d]pyrimidin-6-yl)- 0.1% propyl]-(4-methyl- diethylaminebenzoyl)-amino]- propyl)-carbamic acid tert-butyl ester

Method 23 and Example C-1 (+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]-pyrimidin-6-yl)-propyl]-4-methyl-benzamideHydrogen Chloride

A solution of (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 22) (23 mg, 0.04 mmol) in 3 ml of 4 M HClin dioxane was stirred at room temperature for 2 hr. The solvent wasdistilled off by vacuo, the residue was dried at 40-50° C. for overnightunder vacuum. The corresponding amine chloride salt was obtained. Yieldwas 19 mg (93%). m/z 474 (MH⁺) ¹H NMR (500 MHz, 100° C., DMSO-d₆): 0.68(t, 3H), 1.52 (m, 1H), 1.72 (m, 1H), 1.92 (m, 1H), 2.10 (m, 1H), 2.39(s, 3H), 2.51 (m, 2H), 2.57 (s, 3H), 3.41 (m, 2H), 4.85 (br, 1H), 5.50(br, 1H), 5.77 (d, 1H), 7.07 (br, 2H), 7.24-7.35 (m, 7H), 7.73 (br, 3H).

Method 24 N-(4-Cyano-3-methyl-isothiazol-5-yl)-3-methyl-butyramide

To a solution of 5-amino-3-methylisothiazole-4-carbonitrile (method 4)(6.38 g, 45.9 mmol) in pyridine (20 mL) at 0° C., isovaleryl chloride(6.65 g, 55 mmol) was added dropwise. After the completion of theaddition the reaction mixture was allowed to warm to r.t. and stirredovernight. The TLC and the MS showed the complete disappearance of thestarting material and the reaction mixture was diluted with CHCl₃ (200mL), washed with water (200 mL), 2N HCl (225 mL), satd. NaHCO₃ (200 mL),brine (200 mL) and dried over Na₂SO₄. Concentration of the CHCl₃ layerprovided the crude product which was triturated from DCM/hexanes (1/10)and filtered off to isolateN-(4-cyano-3-methyl-isothiazol-5-yl)-3-methyl-butyramide (8.1 g, 79%) asan off-white crystalline solid. ¹H NMR (300 MHz) δ 1.04 (d, 6H),2.18-2.32 (m, 1H), 2.46 (d, 2H), 2.53 (s, 3H), 9.87 (bs, 1H).

Method 25 3-Methyl-5-(3-methyl-butyrylamino)-isothiazole-4-carboxylicAcid Amide

To a solution ofN-(4-cyano-3-methyl-isothiazol-5-yl)-3-methyl-butyramide (method 24) (8g, 35.8 mmol) in 30% aqueous NH₄OH (200 mL), was added dropwise 100 mLof hydrogen peroxide at r.t. After the completion of the addition thereaction mixture was stirred at 60° C. overnight after which the TLCshowed the complete disappearance of SM. The reaction mixture wasconcentrated to 40 mL and extracted with chloroform (3×100 mL). Theorganic layer was dried (Na₂SO₄) and concentrated to obtain3-methyl-5-(3-methyl-butyrylamino)-isothiazole-4-carboxylic acid amide(6.1 g, 71%) as a light yellow solid. ¹H NMR (300 MHz) δ 1.03 (d, 6H),2.24 (m, 1H), 2.43 (d, 2H), 2.69 (s, 3H), 5.98 (bs, 2H), 11.77 (bs, 1H).

Method 26 6-Isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one

3-Methyl-5-(3-methyl-butyrylamino)-isothiazole-4-carboxylic acid amide(method 25) (6 g, 25 mmol) was suspended in 150 mL of 30% NH₃ and thenwas heated to 140° C. for 5 h in a pressure reactor. The mixture wascooled and neutralized to pH 7. The reaction mixture was extracted withEtOAc (3×100 mL) and the combined organic layers were washed with water(100 mL), brine (100 mL) and concentrated to get the crude product whichwas further purified by column (silica gel) chromatography using 30%EtOAc in hexanes as eluent. Concentration of the pure product fractionsprovided 6-isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one (2.2g, 38%) as an off-white powder. ¹H NMR (300 MHz) δ 1.05 (d, 6H), 2.32(m, 1H), 2.69 (d, 2H), 2.82 (s, 3H).

Method 275-Benzyl-6-isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one

To a solution of6-isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one (method 26)(1.31 g, 5.8 mmol) in 20 mL of anhydrous DMF was added 1.38 g (10 mmol)of anhydrous K₂CO₃ followed by benzyl bromide (118 g, 6.9 mmol) and themixture was stirred at room temperature overnight. The TLC of thereaction mixture showed the complete disappearance of the SM. Thereaction mixture was poured into ice-cold water and extracted with EtOAc(3×100 mL). The combined extracts were washed with water (100 mL), brine(100 mL), dried (Na₂SO₄) and concentrated. The TLC and the ¹H NMR showedthe presence of two products N alkylated as well as O-alkylated productsin a ratio of 7:3. The products were separated by column (silica gel,116 g) chromatography using 10% EtOAc in hexanes.5-Benzyl-6-isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one wasisolated as white crystalline solid (1.3 g, 70%). m/z 314 (MH⁺), ¹H NMR(300 MHz) δ 0.94 (d, 6H), 2.23-2.37 (m, 1H), 2.64 (d, 2H), 2.82 (s, 3H),5.38 (s, 2H), 7.10-7.38 (m, 5H).

Methods 27a-b

The following compounds were synthesized according to Method 27:

Method Alkylating # Compound Name m/z agent 27a5-(4-Fluoro-benzyl)-6-isobutyl- 332 (MH⁺) 4-fluorobenzyl3-methyl-5H-isothiazolo[5,4- bromide d]pyrimidin-4-one 27b5-(3-Fluoro-benzyl)-6-isobutyl- 332 (MH⁺) 3-fluorobenzyl3-methyl-5H-isothiazolo[5,4- bromide d]pyrimidin-4-one

Method 285-Benzyl-6-(1-bromo-2-methyl-propyl)-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one

To a solution of5-benzyl-6-isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 27) (1.3 g, 4.2 mmol) and sodium acetate (2 g) in acetic acid(10 mL) at 100° C., a solution of the bromine (1.32 g, 8.4 mmol) inacetic acid (10 mL) was added dropwise over a period of 20 minutes. Thereaction mixture was stirred at that temperature for 30 min and cooledand the TLC (eluent 10% EtOAc in hexanes) and MS showed the completedisappearance of the SM and only the product. The reaction mixture waspoured into ice water and extracted with EtOAc (3×60 mL) and the organiclayers were combined and washed with 2% sodium thiosulfate solution (60mL), water (100 mL), brine (100 mL) and dried over Na₂SO₄. Concentrationof the organic layer provided5-benzyl-6-(1-bromo-2-methyl-propyl)-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(1.61 g, 99%) as white crystalline solid. m/z 392, 394 (MH⁺), ¹H NMR(300 MHz) δ 0.54 (d, 3H), 1.11 (d, 3H), 2.62-2.76 (m, 1H), 2.83 (s, 3H),4.42 (d, 1H), 4.80 (d, 1H), 6.22 (d, 1H), 7.12-7.42 (m, 5H).

Methods 28a-b

The following compounds were synthesized according to Method 28:

Method # Compound Name m/z SM 28a 6-(1-Bromo-2-methyl-propyl)-5- 410,412 (MH⁺) Method (4-fluoro-benzyl)-3-methyl-5H- 27aisothiazolo[5,4-d]pyrimidin-4-one 28b 6-(1-Bromo-2-methyl-propyl)-5-410, 412 (MH⁺) Method (3-fluoro-benzyl)-3-methyl-5H- 27bisothiazolo[5,4-d]pyrimidin-4-one

Method 296-(1-Azido-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one

To a solution of5-benzyl-6-(1-bromo-2-methyl-propyl)-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 28) (0.6 g, 1.52 mmol) in anhydrous DMF (20 mL), sodium azide(0.65 g, 10 mmol) was added and the mixture was stirred at roomtemperature for 1 hour. The TLC of the RM showed the completedisappearance of the starting bromide. The reaction mixture was pouredinto ice water (300 mL) and extracted with EtOAc (3×100 mL). The organiclayer was washed with water (100 mL), brine (100 mL) and dried (Na₂SO₄).Concentration of the organic layer provided the crude product which waspurified by column (silica gel) chromatography using 30% EtOAc inhexanes as eluent to isolate6-(1-azido-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(0.506 g, 94%) as a low melting solid. m/z 355 (MH⁺), ¹H NMR (300 MHz) δ0.57 (d, 3H), 1.07 (d, 3H), 2.50-2.74 (m, 1H), 2.98 (s, 3H), 3.71 (d,1H), 5.05 (d, 1H), 5.78 (d, 1H), 7.12-7.40 (m, 5H).

Methods 29a-b

The following compounds were synthesized according to Method 29:

Method # Compound Name m/z SM 29a6-(1-Azido-2-methyl-propyl)-5-(4-fluoro- 373 Methodbenzyl)-3-methyl-5H-isothiazolo[5,4- (MH⁺) 28a d]pyrimidin-4-one 29b6-(1-Azido-2-methyl-propyl)-5-(3-fluoro- 373 Methodbenzyl)-3-methyl-5H-isothiazolo[5,4- (MH⁺) 28b d]pyrimidin-4-one

Method 306-(1-Amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one

To a solution of6-(1-azido-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 29) (0.5 g, 1.41 mmol) in methanol (20 mL) was added 5% Pd/C(20% by wt.) and the resulting mixture was stirred at r.t. in anatmosphere of H₂ and the progress of the reaction was monitored by MS.After the disappearance of the starting material the reaction mixturewas filtered through celite and washed with EtOAc. Concentration of thefiltrate provided6-(1-amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-oneas a thick oil. The product was used as such in the next reaction without further purification. m/z 349 (MH⁺)

Methods 30a-b

The following compounds were synthesized according to Method 30:

Method # Compound Name m/z SM 30a6-(1-Amino-2-methyl-propyl)-5-(4-fluoro- 367 Methodbenzyl)-3-methyl-5H-isothiazolo[5,4- (MH⁺) 29a d]pyrimidin-4-one 30b6-(1-Amino-2-methyl-propyl)-5-(3-fluoro- 367 Methodbenzyl)-3-methyl-5H-isothiazolo[5,4- (MH⁺) 29b d]pyrimidin-4-one

Method 31{3-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propylamino]-propyl}-carbamicAcid Tert-Butyl Ester

To a solution of6-(1-amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 30) in DCM (30 mL), 4 Å molecular sieves (5 g) was addedfollowed by (3-oxo-propyl)-carbamic acid tert-butyl ester (1.2 eq) andthe reaction mixture was stirred at r.t. for 3 h and the progress of thereaction was monitored by MS. After the complete disappearance of thestarting amine, a catalytic amount of acetic acid was added to thereaction followed by sodium triacetoxyborohydride (1.2 eq) and thereaction mixture was stirred at r.t. overnight. After the completion ofthe reaction (MS), the reaction mixture was filtered and the residue waswashed with DCM and the filtrate was washed with water (100 mL), brine(100 mL) and concentrated to get the crude product which was used assuch for the next reaction. m/z 486 (MH⁺).

Methods 31a-c

The following compounds were synthesized according to Method 31:

Method # Compound Name m/z SM 31a(3-{1-[5-(4-Fluoro-benzyl)-3-methyl-4-oxo- 504 Method4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6- (MH⁺) 30ayl]-2-methyl-propylamino}-propyl)- carbamic acid tert-butyl ester 31b(3-{1-[5-(3-Fluoro-benzyl)-3-methyl-4-oxo- 504 Method4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6- (MH⁺) 30byl]-2-methyl-propylamino}-propyl)- carbamic acid tert-butyl ester 31c{2-[1-(5-Benzyl-3-methyl-4-oxo-4,5- 472 Methoddihydro-isothiazolo[5,4-d]pyrimidin-6-yl)- (MH⁺) 302-methyl-propylamino]-ethyl}- carbamicacid tert-butyl ester

Method 325-Benzyl-6-[1-(2-[1,3]dioxolan-2-yl-ethylamino)-2-methyl-propyl]-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one

To a solution of6-(1-amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 30) (1.6 g, 4.88 mmol) in anhydrous DMF (20 mL),2-(2-bromo-ethyl)-[1,3]dioxolane (0.88 g, 4.88 mmol) was added and theresulting solution was heated at 70° C. for 2 h. The reaction mixturewas cooled, diluted with water and extracted with EtOAc (3×60 mL). Thecombined organic extracts were dried (Na₂SO₄) and concentrated toprovide the crude product (2 g), which was used as such in the nextreaction. m/z 429 (MH⁺); ¹H-NMR (300 MHz) δ 0.88 (d, 3H), 0.96 (d, 3H),1.54-1.62 (m, 2H), 1.86-2.05 (m, 2H), 2.18 (bs, 1H), 2.38-2.46 (m, 1H),2.84 (s, 3H), 3.57 (d, 1H), 3.74-3.94 (m, 4H), 4.78 (t, 1H), 4.99 (d,1H), 5.85 (d, 1H), 7.15-7.38 (m, 5H).

Method 33{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicAcid Tert-Butyl Ester

To a solution of the crude{3-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propylamino]-propyl}-carbamicacid tert-butyl ester (method 31) in pyridine (10 mL) at r.t., asolution of the p-toluoyl chloride (0.616 g, 4 mmol) in DCM (10 mL) wasadded dropwise and the resulting solution was stirred at r.t. for 2days. The reaction mixture was diluted with DCM (100 mL) washed withwater (2×100 mL), brine (100 mL) and dried (Na₂SO₄). Concentration ofthe organic layer provided the crude product which was purified bycolumn (silica gel) chromatography using 20-30% EtOAc in hexanes aseluent. Product isolated was 0.276 g. m/z 604 (MH⁺).

Methods 33a-2

The following compounds were synthesized according to Method 33:

Method # Compound Name m/z SM Acylating agent 33a{3-[{1-[5-(4-Fluoro-benzyl)-3-methyl-4- 622 (MH⁺) Method4-methyl-benzoyl oxo-4,5-dihydro-isothiazolo[5,4- 31a chlorided]pyrimidin-6-yl]-2-methyl-propyl}-(4- methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester 33b{3-[{1-[5-(3-Fluoro-benzyl)-3-methyl-4- 622 (MH⁺) Method4-methyl-benzoyl oxo-4,5-dihydro-isothiazolo[5,4- 31b chlorided]pyrimidin-6-yl]-2-methyl-propyl}-(4- methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester 33c {2-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-590 (MH⁺) Method 4-methyl-benzoyl dihydro-isothiazolo[5,4-d]pyrimidin-6-31c chloride yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-ethyl}-carbamic acid tert-butyl ester 33d{2-[[1-(5-Benzyl-3-methyl-4-oxo-4,5- 654, Method 4-bromo-benzoyldihydro-isothiazolo[5,4-d]pyrimidin-6- 656 (MH⁺) 31c chlorideyl)-2-methyl-propyl]-(4-bromo-benzoyl)- amino]-ethyl}-carbamic acidtert-butyl ester 33e {2-[[1-(5-Benzyl-3-methyl-4-oxo-4,5- 608 (MH⁺)Method 3-fluoro-4- dihydro-isothiazolo[5,4-d]pyrimidin-6- 31cmethyl-benzoyl yl)-2-methyl-propyl]-(3-fluoro-4-methyl- chloridebenzoyl)-amino]-ethyl}-carbamic acid tert-butyl ester 33f{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5- 622 (MH⁺) Method 3-fluoro-4-dihydro-isothiazolo[5,4-d]pyrimidin-6- 31 methyl-benzoylyl)-2-methyl-propyl]-(3-fluoro-4-methyl- chloridebenzoyl)-amino]-propyl}-carbamic acid tert-butyl ester 33g{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5- 668, Method 4-bromo-benzoyldihydro-isothiazolo[5,4-d]pyrimidin-6- 670 (MH⁺) 31 chlorideyl)-2-methyl-propyl]-(4-bromo-benzoyl)- amino]-propyl}-carbamic acidtert-butyl ester

Methods 34a-2

The following compounds were chirally purified in same manner as (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 12). Chiral purification generallyresulted in 99% purity of the (+) enantiomer.

Method Column Solvent (+) Enantiomer # Compound Name Type compositionretention time SM 34a (+) {3-[{1-[5-(4-Fluoro- Chiralpak 85% hexanes 7.1min Method benzyl)-3-methyl-4-oxo- AD 15% 33a 4,5-dihydro- isopropanolisothiazolo[5,4- 0.1% d]pyrimidin-6-yl]-2- diethylaminemethyl-propyl}-(4- methyl-benzoyl)-amino]- propyl}-carbamic acidtert-butyl ester 34b (+) {3-[{1-[5-(3-Fluoro- Chiralpak 85% hexanes 8.0min Method benzyl)-3-methyl-4-oxo- AD 15% 33b 4,5-dihydro- isopropanolisothiazolo[5,4- 0.1% d]pyrimidin-6-yl]-2- diethylaminemethyl-propyl}-(4- methyl-benzoyl)-amino]- propyl}-carbamic acidtert-butyl ester 34c (+) {2-[[1-(5-Benzyl-3- Chiralpak 80% hexanes 7.7min Method methyl-4-oxo-4,5- AD 20% 33c dihydro-isothiazolo[5,4-isopropanol d]pyrimidin-6-yl)-2- 0.1% methyl-propyl]-(4- diethylaminemethyl-benzoyl)-amino]- ethyl}-carbamic acid tert- butyl ester 34d (+){2-[[1-(5-Benzyl-3- Chiralpak 75% hexanes 7.9 min Methodmethyl-4-oxo-4,5- AD 25% 33d dihydro-isothiazolo[5,4- isopropanold]pyrimidin-6-yl)-2- 0.1% methyl-propyl]-(4- diethylaminebromo-benzoyl)-amino]- ethyl}-carbamic acid tert- butyl ester 34e (+){2-[[1-(5-Benzyl-3- Chiralpak 75% hexanes 6.3 min Methodmethyl-4-oxo-4,5- AD 25% 33e dihydro-isothiazolo[5,4- isopropanold]pyrimidin-6-yl)-2- 0.1% methyl-propyl]-(3-fluoro- diethylamine4-methyl-benzoyl)- amino]-ethyl}-carbamic acid tert-butyl ester 34f (+){3-[[1-(5-Benzyl-3- Chiralpak 80% hexanes 8.6 min Methodmethyl-4-oxo-4,5- AD 20% 33f dihydro-isothiazolo[5,4- isopropanold]pyrimidin-6-yl)-2- 0.1% methyl-propyl]-(3-fluoro- diethylamine4-methyl-benzoyl)- amino]-propyl}-carbamic acid tert-butyl ester 34g (+){3-[[1-(5-Benzyl-3- Chiralpak 80% hexanes 7.0 min Methodmethyl-4-oxo-4,5- AD 20% 33g dihydro-isothiazolo[5,4- isopropanold]pyrimidin-6-yl)-2- 0.1% methyl-propyl]-(4- diethylaminebromo-benzoyl)-amino]- propyl}-carbamic acid tert-butyl ester Chiralpurification generally resulted in 99% purity of the (+) enantiomer.

Method 35N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamideHydrogen Chloride

{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicacid tert-butyl ester (method 33) (0.245 g, 0.40 mmol) was dissolved in4M HCl in 1,4-dioxane and the mixture was stirred at r.t. for 20 min andthe TLC showed the complete disappearance of the starting material. Thereaction mixture was concentrated in a rotary evaporator and the residuewas triturated with ether. The precipitated product was filtered off andwashed with ether and dried under vacuo to yieldN-(3-amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamideas the hydrochloride salt (0.219 g, 100%). White powder, mp. 139-140° C.m/z 504 (MH⁺), ¹H NMR (DMSO-d₆, 300 MHz, 96° C.) δ: 0.45 (d, 3H), 0.90(d, 3H), 1.12-1.30 (m, 1H), 1.46-1.63 (m, 1H), 2.25 (t, 2H), 2.36 (s,3H), 2.64-2.7 (m, 1H), 2.68 (s, 3H), 3.34 (t, 2H), 5.06 (d, 1H), 5.59(d, 1H), 5.90 (d, 1H), 7.20-7.40 (m, 9H), 7.71 (bs, 3H).

Methods 35a-2

The following compounds were synthesized according to Method 35:

Method # Compound Name m/z SM 35a(+)N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-benzyl)-3-methyl- 522 (MH⁺)Method 4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl- 34apropyl}-4-methyl-benzamide hydrogen chloride Example D-1 35b(+)N-(3-Amino-propyl)-N-{1-[5-(3-fluoro-benzyl)-3-methyl- 522 (MH⁺)Method 4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl]-2-methyl- 34bpropyl}-4-methyl-benzamide hydrogen chloride Example D-3 35c(+)N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5- 490 (MH⁺) Methoddihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4- 34cmethyl-benzamide hydrogen chloride Example D-5 35d(+)N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5- 554, Methoddihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4- 556 (MH⁺)34d bromo-benzamide hydrogen chloride Example D-4 35e(+)N-(2-Amino-ethyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5- 508 (MH⁺) Methoddihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3- 34efluoro-4-methyl-benzamide hydrogen chloride Example D-6 35f(+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5- 522 (MH⁺)Method dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-3- 34ffluoro-4-methyl-benzamide hydrogen chloride Example D-7 35g(+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5- 568, Methoddihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4- 570 (MH⁺)34g bromo-benzamide hydrogen chloride Example D-8

Method 36 and Example D-2 Chiral purification of (+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamide

The following compound was chirally purified in same manner as (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 12). Chiral purification generallyresulted in 99% purity of the (+) enantiomer.

Method Column Solvent (+) Enantiomer # Compound Name Type compositionretention time SM 36 (+) N-(3-Amino- Chiralpak 70% hexanes 8.0 minMethod propyl)-N-[1-(5-benzyl- AD 30% 35 3-methyl-4-oxo-4,5- isopropanoldihydro-isothiazolo[5,4- 0.1% d]pyrimidin-6-yl)-2- diethylaminemethyl-propyl]-4- methyl-benzamide Example D-2

Method 37N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-N-(2-[1,3]dioxolan-2-yl-ethyl)-benzamide

5-Benzyl-6-[1-(2-[1,3]dioxolan-2-yl-ethylamino)-2-methyl-propyl]-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 32) (1 g, 2.33 mmol) was dissolved in chloroform (70 mL) and tothe chloroform solution diisopropylethyl amine (0.9 g, 6.99 mmol) wasadded followed by the addition of 4-bromobenzoyl chloride (0.76 g, 3.49mmol) and the mixture was refluxed overnight. The MS showed thedisappearance of the starting material and only the product peak at 611(MH⁺). The reaction mixture was concentrated and column purified (silicagel, 160 g) using 10-20% EtOAc in hexanes as eluent. The concentrationof the product fractions provided the pure product as white foam (1.1 g,77%). m/z 611, 613 (MH⁺); ¹H-NMR (300 MHz) δ 0.35 (d, 3H), 0.94 (d, 3H),0.94-1.06 (m, 1H), 1.36-1.46 (m, 1H), 2.68-2.78 (m, 1H), 2.88 (s, 3H),3.38-3.52 (m, 1H), 3.54-3.70 (m, 5H), 4.34 (t, 1H), 5.18 (d, 1H), 5.73(d, 1H), 6.13 (d, 1H), 7.20 (d, 2H), 7.26-7.46 (m, 5H), 7.56 (d, 2H).

Methods 37a-b

The following compounds were synthesized according to Method 37:

Acylating Method # Compound Name m/z SM agent 37aN-[1-(5-Benzyl-3-methyl-4-oxo-4,5- 547 (MH⁺) Method 4-methyl-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)- 32 benzoyl2-methyl-propyl]-N-(2-[1,3]dioxolan-2-yl- chlorideethyl)-4-methyl-benzamide 37b N-[1-(5-Benzyl-3-methyl-4-oxo-4,5- 565(MH⁺) Method 3-fluoro-4- dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)- 32methyl- 2-methyl-propyl]-N-(2-[1,3]dioxolan-2-yl- benzoylethyl)-3-fluoro-4-methyl-benzamide chloride

Method 38N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-N-(3-oxo-propyl)-benzamide

N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-N-(2-[1,3]dioxolan-2-yl-ethyl)-benzamide(method 37) (1.1 g, 1.8 mmol) was dissolved in 20 mL of 80% acetic acidand the solution was heated at 80° C. for 2 h. The reaction mixture wascooled in an ice bath and neutralized slowly by the addition of solidNaHCO₃ until pH 8. The thus obtained mixture was extracted with DCM(3×100 mL). The combined organic layers was washed with brine (100 mL)and dried (Na₂SO₄). Concentration of the DCM layer provided a yellowfoam (1 g crude yield) and it was used as such in the next reaction. m/z567, 569 (MH⁺)

Methods 38a-b

The following compounds were synthesized according to Method 38:

Method # Compound Name m/z SM 38a N-[1-(5-Benzyl-3-methyl-4-oxo-4,5- 503Method 37a dihydro-isothiazolo[5,4-d]pyrimidin- (MH⁺)6-yl)-2-methyl-propyl]-4-methyl-N- (3-oxo-propyl)-benzamide 38bN-[1-(5-Benzyl-3-methyl-4-oxo-4,5- 521 Method 37bdihydro-isothiazolo[5,4-d]pyrimidin- (MH⁺)6-yl)-2-methyl-propyl]-3-fluoro-4- methyl-N-(3-oxo-propyl)-benzamide

Method 39N-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-N-(3-dimethylamino-propyl)-benzamide

To a solution ofN-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-bromo-N-(3-oxo-propyl)-benzamide(method 38) (1 g, 1.76 mmol) in methanol (20 mL) two drops of aceticacid were added followed by the addition of dimethylamine (1 mL, 2Msolution in THF) and sodium cyanoborohydride (0.314 g, 5 mmol) and themixture was stirred at room temperature for 3 h. The reaction mixturewas concentrated and the residue was dissolved in DCM (100 mL) and theorganic layer was washed with satd. NaHCO₃ (3×100 mL). The organic layerwas concentrated and the crude product was purified by columnchromatography using 0-10% MeOH in EtOAc. The pure product fractionswere concentrated and the thus obtained foam was crystallized fromether/hexanes to get the product as white crystalline solid. Yield was0.366 g (35%). m/z 596, 598 (MH⁺); ¹H-NMR (300 MHz) δ 0.35 (d, 3H),0.66-0.77 (m, 1H), 0.93 (d, 3H), 0.18-1.27 (m, 1H), 1.65-1.85 (m, 2H),1.80 (s, 6H), 2.66-2.76 (m, 1H), 2.89 (s, 3H), 3.30-3.41 (m, 2H), 5.20(d, 1H), 5.73 (d, 1H), 6.15 (d, 1H), 7.20 (d, 2H), 7.28-7.41 (m, 5H),7.56b (d, 2H).

Methods 39a-b

The following compounds were synthesized according to Method 39:

Method # Compound Name m/z SM 39a N-[1-(5-Benzyl-3-methyl-4-oxo- 532(MH⁺) Method 38a 4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl- propyl]-N-(3-dimethylamino-propyl)-4-methyl-benzamide 39b N-[1-(5-Benzyl-3-methyl-4-oxo- 540 (MH⁺)Method 38b 4,5-dihydro-isothiazolo[5,4- d]pyrimidin-6-yl)-2-methyl-propyl]-N-(3-dimethylamino- propyl)-3-fluoro-4-methyl- benzamide

Methods 40-40b

The following compounds were chirally purified in same manner as (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 12). Chiral purification generallyresulted in 99% purity of the (+) enantiomer.

Method Column Solvent (+) Enantiomer # Compound Name Type compositionretention time SM 40 (+) N-[1-(5-Benzyl-3- Chiralpak 85% hexanes 7.6 minMethod methyl-4-oxo-4,5- AD 15% 39 dihydro-isothiazolo[5,4- isopropanold]pyrimidin-6-yl)-2- 0.1% methyl-propyl]-4-bromo- diethylamineN-(3-dimethylamino- propyl)-benzamide Example E-2 40a (+)N-[1-(5-Benzyl-3- Chiralpak 90% hexanes 7.7 min Method methyl-4-oxo-4,5-AD 10% 39a dihydro-isothiazolo[5,4- isopropanol d]pyrimidin-6-yl)-2-0.1% methyl-propyl]-N-(3- diethylamine dimethylamino-propyl)-4-methyl-benzamide Example E-1 40b (+) N-[1-(5-Benzyl-3- Chiralpak 90%hexanes 7.5 min Method methyl-4-oxo-4,5- AD 10% 39bdihydro-isothiazolo[5,4- isopropanol d]pyrimidin-6-yl)-2- 0.1%methyl-propyl]-N-(3- diethylamine dimethylamino-propyl)-3-fluoro-4-methyl- benzamide Example E-3

Method 41 3-Methyl-5-(3-methyl-butyryl)-isoxazole-4-carboxylic AcidAmide

A mixture of 5-amino-3-methyl-isoxazole-4-carboxylic acid amide (10 g,70 mmol) in 25 ml of isovaleric anhydride was stirred at 110-145° C. for1 h. The brown solution was diluted with hexane (500 ml) and cooleddown. The precipitated gum was separated from the mixture and washedwith hexane, dried in vacuo.3-Methyl-5-(3-methyl-butyryl)-isoxazole-4-carboxylic acid amide wasobtained as a yellow gum. Further used without purification in method42.

Method 42 6-Isobutyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one

A suspension of 3-methyl-5-(3-methyl-butyryl)-isoxazole-4-carboxylicacid amide (method 41) (split into 40 vials) in 3.5 ml of 2N NaOH aq wassubjected to microwave irradiation at 140° C. for 20 min. The resultingsolution was cooled with an ice bath, and the pH was adjusted to 13 withconcentrated HCl. The solid was filtered, washed with water, dried overvacuum at 40° C. overnight.6-Isobutyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one (8 g) wasobtained as a white solid. 55% yield for two steps. m/z: 208 (MH⁺), ¹HNMR (DMSO-d₆): 0.76 (d, 6H), 1.95 (m, 1H), 2.25 (s, 3H), 2.32 (d, 2H),12.55 (s, 1H).

Method 435-Benzyl-6-isobutyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one

A suspension of 6-isobutyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one(method 42) (5 g, 24.4 mmol), benzylbromide (4.17 g, 24.4 mmol),potassium carbonate (6.7 g, 48.8 mmol) in 20 ml DMF was stirred at roomtemperature for 2 days. The mixture was diluted with water, extractedwith EtOAc (100 ml×3), the combined organic phases were dried,concentrated, purified by flash column chromatography (elute:hexane-EtOAc=7:1).5-benzyl-6-isobutyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one wasobtained as white solid (3 g, 10.1 mmol) (41%). m/z: 298 (MH⁺), ¹H NMR(DMSO-d₆): 0.90 (d, 6H), 2.30 (m, 1H), 2.55 (s, 3H), 2.75 (d, 2H), 5.42(s, 2H), 7.22-7.43 (m, 5H).

Methods 43a-b

The following compounds were synthesized according to Method 43:

Method # Compound Name m/z 43a5-(4-Fluoro-benzyl)-6-isobutyl-3-methyl-5H- 316 (MH⁺)isoxazolo[5,4-d]pyrimidin-4-one 43b5-(3-Fluoro-benzyl)-6-isobutyl-3-methyl-5H- 316 (MH⁺)isoxazolo[5,4-d]pyrimidin-4-one

Method 445-Benzyl-6-(1-bromo-2-methyl-propyl)-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one

A solution of5-benzyl-6-isobutyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one (method43) (130 mg, 0.44 mmol) and sodium acetate (90 mg, 1.09 mmol, 2.5 eq) inglacial acetic acid (2 ml) was treated with a preformed bromine solution(0.7 ml bromine in 10 ml of glacial acetic acid) (1.54 ml, 2 mmol). Themixture was stirred at 110-120° C. for 1 day. Excess bromine (1.54 ml, 2mmol) was added to the mixture every 4 hours for two times at 110-120°C. Water was added to the mixture to which was subsequently addedpotassium carbonate and extracted with DCM (20 ml×3), the combinedorganic phases were washed with water and dried, then concentrated togive the crude product which was purified by ISCO (elute: hexane-EtOAc).100 mg (60%) of5-benzyl-6-(1-bromo-2-methyl-propyl)-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-onewas obtained as a yellow gum. m/z: 376, 378 (MH⁺), ¹H NMR (DMSO-d₆):0.55 (d, 3H), 1.02 (d, 3H), 2.48 (m, 4H), 4.75 (d, 1H), 5.60 (d, 1H),5.70 (d, 1H), 7.16-7.30 (m, 5H).

Methods 44a-b

The following compounds were synthesized according to Method 44:

Method # Compound Name m/z SM 44a 6-(1-Bromo-2-methyl-propyl)-5-(4- 394,396 Method fluoro-benzyl)-3-methyl-5H- (MH⁺) 43aisoxazolo[5,4-d]pyrimidin-4-one 44b 6-(1-Bromo-2-methyl-propyl)-5-(3-394, 396 Method fluoro-benzyl)-3-methyl-5H- (MH⁺) 43bisoxazolo[5,4-d]pyrimidin-4-one

Method 456-(1-Azido-2-methyl-propyl)-5-benzyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one

A suspension of5-benzyl-6-(1-bromo-2-methyl-propyl)-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one(method 44) (100 mg, 0.266 mmol) and sodium azide (34.5 mg, 0.53 mmol)in DMF (2 ml) was stirred at 60° C. for 1 h. Water (5 ml) was added tothe mixture and then extracted with EtOAc (3×20 ml). The combinedorganic phases were washed with brine (10 ml), dried, concentrated toobtain6-(1-azido-2-methyl-propyl)-5-benzyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-onewhich was purified by ISCO (Hexane-EtOAc). 50 mg (56%) of a colorlessoil was obtained. m/z: 339 (MH⁺), ¹H NMR (DMSO-d₆): 0.60 (d, 3H), 0.95(d, 3H), 2.25 (m, 1H), 2.45 (s, 3H), 4.19 (d, 1H), 5.30 (d, 1H), 5.42(d, 1H), 7.12-7.30 (m, 5H).

Methods 45a-b

The following compounds were synthesized according to Method 45:

Method # Compound Name m/z SM 45a 6-(1-Azido-2-methyl-propyl)-5-(4- 357(MH⁺) Method fluoro-benzyl)-3-methyl-5H- 44aisoxazolo[5,4-d]pyrimidin-4-one 45b 6-(1-Azido-2-methyl-propyl)-5-(3-357 (MH⁺) Method fluoro-benzyl)-3-methyl-5H- 44bisoxazolo[5,4-d]pyrimidin-4-one

Method 466-(1-Amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one

A mixture of6-(1-azido-2-methyl-propyl)-5-benzyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one(method 45) (40 mg, 1.118 mmol), triphenylphosphine (62 mg, 0.237 mmol)and water (4 μl) in THF was stirred at 60° C. for 5 hours. Excess amountof water (30 μl) was added to the mixture and stirred at 60° C. foranother 10 hours. The volatile solvent was distilled out, the crudeproduct was purified by ISCO (EtOAc:hexane=60%. 25 mg (68%) of6-(1-amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-onewas obtained as colorless oil. m/z: 313 (MH⁺), ¹H NMR (DMSO-d₆): 0.55(d, 3H), 0.95 (d, 3H), 2.02 (m, 1H), 2.15 (br, 2H), 2.55 (s, 3H), 3.59(d, 1H), 5.38 (d, 1H), 5.65 (d, 1H), 7.25-7.42 (m, 5H).

Methods 46a-b

The following compounds were synthesized according to Method 46:

Method # Compound Name m/z SM 46a 6-(1-Amino-2-methyl-propyl)-5- 331(MH⁺) Method (4-fluoro-benzyl)-3-methyl-5H- 45aisoxazolo[5,4-d]pyrimidin-4-one 46b 6-(1-Amino-2-methyl-propyl)-5- 331(MH⁺) Method (3-fluoro-benzyl)-3-methyl-5H- 45bisoxazolo[5,4-d]pyrimidin-4-one

Method 47{3-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propylamino]-propyl}-carbamicAcid Tert-Butyl Ester

A mixture of6-(1-amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isoxazolo[5,4-d]pyrimidin-4-one(method 46) (20 mg, 0.064 mmol) and (3-oxo-propyl)-carbamic acidtert-butyl ester (11 mg, 0.064 mmol) in DCM (5 ml) with dried 4 ÅMS wasstirred for 1 h at room temperature. Then sodium triacetoxyborohydride(2 eq) and 1 drop of acetic acid were added to the mixture. The mixturewas stirred at room temperature for 1 day. The mixture was filteredthrough a 2μ cartridge, the filtrate was concentrated, the crude mixturewas purified by ISCO (elute: EtOAc-hexane=30%˜60%) to give 18 mg (60%)of{3-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propylamino]-propyl}-carbamicacid tert-butyl ester as a white solid. m/z: 470 (MH⁺), ¹H NMR(DMSO-d₆): 0.65 (d, 3H), 0.80 (d, 3H), 1.10 (m, 2H), 1.25 (s, 9H), 1.32(d, 1H), 1.70-1.90 (m, 2H), 2.18 (m, 1H), 2.49 (s, 3H), 2.70 (m, 2H),3.48 (d, 1H), 5.15 (d, 1H), 5.51 (d, 1H), 6.55 (br, 1H), 7.12-7.32 (m,5H).

Methods 47a-b

The following compounds were synthesized according to Method 47:

Method # Compound Name m/z SM 47a (3-{1-[5-(4-Fluoro-benzyl)-3-methyl-488 (MH⁺) Method 4-oxo-4,5-dihydro-isoxazolo[5,4- 46ad]pyrimidin-6-yl]-2-methyl- propylamino}-propyl)-carbamic acidtert-butyl ester 47b (3-{1-[5-(3-Fluoro-benzyl)-3- 488 (MH⁺) Methodmethyl-4-oxo-4,5-dihydro- 46b isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propylamino}- propyl)-carbamic acid tert-butyl ester

Method 48{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicAcid Tert-Butyl Ester

A solution of{3-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propylamino]-propyl}-carbamicacid tert-butyl ester (method 47) (100 mg, 0.213 mmol) in DCM (4 ml) wasadded p-toluoyl chloride (66 mg, 0.426 mmol) followed by triethylamine(65 mg, 0.639 mmol). The mixture was stirred at 30-40° C. for 2 days.The mixture was then diluted with DCM, washed with saturated sodiumbicarbonate aq. The organic phase was dried, filtered, and concentrated.The crude oil was purified by ISCO (solvent: EtOAc-hexane) to give{3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicacid tert-butyl ester as white solid (115 mg, 0.196 mmol). m/z: 588(MH⁺).

Methods 48a-b

The following compounds were synthesized according to Method 48:

Acylating Method # Compound Name m/z SM agent 48a{3-[{1-[5-(4-Fluoro-benzyl)-3-methyl-4-oxo-4,5- 606 (MH⁺) Method4-methyl- dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2- 47a benzoylmethyl-propyl}-(4-methyl-benzoyl)-amino]- chloride propyl}-carbamic acidtert-butyl ester 48b {3-[{1-[5-(3-Fluoro-benzyl)-3-methyl-4-oxo-4,5- 606(MH⁺) Method 4-methyl- dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2- 47bbenzoyl methyl-propyl}-(4-methyl-benzoyl)-amino]- chloridepropyl}-carbamic acid tert-butyl ester

Method 49 Chiral Purification of (+){3-[{1-[5-(3-Fluoro-benzyl)-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-(4-methyl-benzoyl)-amino]-propyl}-carbamicAcid Tert-Butyl Ester

The following compound was chirally purified in same manner as (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 12). Chiral purification generallyresulted in 99% purity of the (+) enantiomer.

Method Column Solvent (+) Enantiomer # Compound Name Type compositionretention time SM 49 (+) {3-[{1-[5-(3-Fluoro- Chiralpak 80% hexane 7.4min Method benzyl)-3-methyl-4-oxo- AD 20% 48b 4,5-dihydro- isopropanolisoxazolo[5,4- 0.1% d]pyrimidin-6-yl]-2- diethylamine methyl-propyl}-(4-methyl-benzoyl)-amino]- propyl}-carbamic acid tert-butyl ester

Method 50N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamideHydrogen Chloride

A solution of{3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicacid tert-butyl ester (method 48) (0.058 g, 0.1 mmol) in 3 ml of 4 M HClin dioxane was stirred at room temperature for 2 hr. The solvent wasdistilled off by vacuo, the residue was dried at 40˜50° C. for overnightunder vacuum.N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isoxazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-4-methyl-benzamidewas obtained as the HCl salt. Yield was 0.046 g (88%). m/z 488 (MH⁺), ¹HNMR (500 MHz, 100° C., DMSO-d₆): 0.48 (d, 3H), 0.94 (d, 3H), 1.30 (m,1H), 1.60 (m, 1H), 2.35 (m, 2H), 2.38 (s, 3H), 2.58 (s, 3H), 2.70 (m,1H), 3.37 (m, 2H), 5.11 (d, 1H), 5.64 (d, 1H), 5.90 (d, 1H), 7.23-7.39(m, 9H), 7.63 (br, 3H).

Methods 50a-b

The following compounds were synthesized according to Method 50:

Method # Compound Name m/z SM 50a N-(3-Amino-propyl)-N-{1-[5-(4-fluoro-506 Method benzyl)-3-methyl-4-oxo-4,5-dihydro- (MH⁺) 48aisoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl- propyl}-4-methyl-benzamidehydrogen chloride 50b (+) N-(3-Amino-propyl)-N-{1-[5- 506 Method(3-fluoro-benzyl)-3-methyl-4-oxo-4,5- (MH⁺) 49dihydro-isoxazolo[5,4-d]pyrimidin-6-yl]-2-methyl-propyl}-4-methyl-benzamide hydrogen chloride Example F-3

Methods 51 and 51a

The following compounds were chirally purified in same manner as (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 12). Chiral purification generallyresulted in 99% purity of the (+) enantiomer.

Method Column Solvent (+) Enantiomer # Compound Name Type compositionretention time SM 51 (+) N-(3-Amino-propyl)- Chiralpak 60% hexanes 7.9min Method N-[1-(5-benzyl-3-methyl- AD 40% 50 4-oxo-4,5-dihydro-isopropanol isoxazolo[5,4- 0.1% d]pyrimidin-6-yl)-2- diethylaminemethyl-propyl]-4-methyl- benzamide Example F-1 51a (+)N-(3-Amino-propyl)- Chiralpak 60% hexanes 7.5 min MethodN-{1-[5-(4-fluoro- AD 40% 50a benzyl)-3-methyl-4-oxo- isopropanol4,5-dihydro- 0.1% isoxazolo[5,4- diethylamine d]pyrimidin-6-yl]-2-methyl-propyl}-4- methyl-benzamide Example F-2

Method 52 3-Amino-2-thioformyl-but-2-enoic Acid Ethyl Ester

To an ice cold solution of phosphoryl chloride (20 mL, 220 mmol),anhydrous DMF (60 mL) was added dropwise and the resulting solution wasadded dropwise during 30 min to a stirred solution of the ethylcrotonate (25.83 g, 200 mmol) in anhydrous THF (400 mL) with thetemperature maintained at 0° C. The resulting mixture was allowed towarm to room temperature and stirred overnight and then for 4 h at 30°C.; it was then allowed to stand overnight in a refrigerator. Additionof ether (200 mL) resulted in a yellow oil from which the ether layerwas decanted. The resulting oil was washed several times with etheruntil the ether layer became clear. The oily product was dissolved inDCM (800 mL) and was vigorously shaken with aqueous sodium hydrogensulfide (2M; 500 mL). The organic layer was separated and the aqueouslayer washed with DCM (100 mL). The combined organic layers were washedwith water (600 mL), brine (400 mL), dried (Na₂SO₄) and concentrated toget orange crystals. The thus obtained product was triturated withDCM/hexanes to get pure product as orange crystals (25.6 g, 74%). ¹H NMR(300 MHz) δ: 1.33 (t, 3H), 2.57 (s, 3H), 4.23 (q, 2H), 6.83 (bs, 1H),10.97 (s, 1H), 13.93 (s, 1H).

Method 53 3-Methyl-isothiazole-4-carboxylic Acid Ethyl Ester

To a solution of 3-amino-2-thioformyl-but-2-enoic acid ethyl ester(method 52) (25.6 g, 147 mmol) in ethanol (300 mL), was addedm-chloroperbenzoic acid (33.3 g, 77%, 149 mmol) in ethanol (200 mL)dropwise with stirring at room temperature. After the completion of theaddition the reaction mixture was heated at 75° C. for 2 h after whichthe MS showed the complete disappearance of the starting material. Thereaction mixture was diluted with ether (500 mL) and the etherealsolution was washed with 0.1 M NaOH solution (3×500 mL) and once withwater (400 mL) dried (Na₂SO₄) and concentrated to get the pure productas light brown oil. Yield 23.5 g (93%). ¹H NMR (300 MHz) δ: 1.40 (t,3H), 2.73 (s, 3H), 5.07 (t, 1H), 4.36 (q, 2H), 9.24 (s, 1H).

Method 54 3-Methyl-isothiazole-4-carboxylic Acid

To a solution of 3-methyl-isothiazole-4-carboxylic acid ethyl ester(method 53) (23.3 g, 136 mmol) in THF (200 mL) aqueous NaOH (6.5 g, 162mmol, in 100 ml of water) was added and the mixture was stirred at roomtemperature for 16 h. The TLC of the reaction mixture showed thecomplete disappearance of the starting material. The reaction mixturewas cooled in an ice bath and acidified to pH 5 using 6M HCl and theresultant mixture was extracted with ether (3×100 mL). The ether layerswere combined, washed with water (100 mL), brine (100 mL), dried(Na₂SO₄) and concentrated to about 10 mL. Addition of hexanes to theabove mixture resulted in the precipitation of the product which wasfiltered off, washed with hexanes and dried to provide the pure productas a tan powder. Yield 15.3 g (79%). ¹H NMR (300 MHz) δ 2.39 (s, 3H),8.98 (s, 1H).

Method 55 (3-Methyl-isothiazol-4-yl)-carbamic Acid Tert-Butyl Ester

To a solution of 3-methyl-isothiazole-4-carboxylic acid (method 54)(14.8 g, 103 mmol) in anhydrous t-BuOH (100 mL) triethyl amine (10.5 g,104 mmol) was added followed by the dropwise addition ofdiphenylphosphoryl azide (28.6 g, 104 mmol) and the resulting mixturewas heated at reflux overnight after which the TLC showed the completedisappearance of the starting material. The reaction mixture was cooledto room temperature and poured into ice cold water (500 mL). The aqueouslayer was extracted with ether (3×100 mL) and the combined organiclayers were washed with satd, NaHCO₃ (100 mL), brine (100 mL) and dried(Na₂SO₄). Concentration of the ether solution provided the crude productwhich was purified by column chromatography to get the pure product aslight brown crystals. Yield 21.4 g (97%). ¹H NMR (300 MHz) δ 1.53 (s,9H), 2.40 (s, 3H), 6.50 (s, 1H), 8.66 (s, 1H).

Method 56 4-tert-Butoxycarbonylamino-3-methyl-isothiazole-5-carboxylicAcid

To a solution of (3-methyl-isothiazol-4-yl)-carbamic acid tert-butylester (method 55) (21.4 g, 100 mmol) in anhydrous THF (200 mL) at −78°C., LDA (139 mL, 1.8 M solution, 250 mmol) was added dropwise over aperiod of 1 h. The reaction mixture was stirred at that temperature fora further 3 h after which powdered dry ice was added and the reactionslowly allowed to warm to room temperature overnight. The reactionmixture was quenched by adding saturated NH₄Cl solution and extractedwith ether (3×100 mL) and the combined ether layers were back extractedwith satd. NaHCO₃ (3×100 mL). The aqueous layers were combined andacidified to pH 5 using 6M HCl and extracted with ether (4×100 mL). Thecombined ether layers were dried (Na₂CO₃) and concentrated to give thepure acid as an off white powder. Yield 11 g (39%). ¹H NMR (300 MHz) δ1.47 (s, 9H), 2.44 (s, 3H), 8.53 (bs, 1H), 9.68 (bs, 1H).

Method 57 4-Amino-3-methyl-isothiazole-5-carboxylic Acid

4-tert-Butoxycarbonylamino-3-methyl-isothiazole-5-carboxylic acid(method 56) (11 g, 45 mmol) was dissolved in 50 mL of 4M solution of HClin 1,4-dioxane (200 mmol) and the resulting solution was stirred at roomtemperature overnight. The TLC showed the complete disappearance of thestarting acid. The reaction was concentrated and the residue wastriturated with ether and the precipitated hydrochloride salt wasfiltered off and washed with ether and dried to provide the product as alight brown powder. Yield 8.2 g (100%). ¹H NMR (300 MHz, DMSO-d₆) δ 2.30(s, 3H), 8.85 (bs, 3H).

Method 58 3-Methyl-5-propyl-isothiazolo[4,5-d][1,3]oxazin-7-one

To a solution of 4-amino-3-methyl-isothiazole-5-carboxylic acid (method57) (2.91 g, 15 mmol) in pyridine (20 mL) at 0° C., was added dropwise asolution of butyryl chloride (3.18 g, 30 mmol) in chloroform (30 mL).The reaction mixture was allowed to warm to room temperature and stirredovernight. Chloroform (200 mL) was added to the reaction mixturefollowed by 2M HCl (200 mL) and the mixture was stirred. The chloroformlayer was further washed with 2M HCl (100 mL), water (100 mL), brine(100 mL) and concentrated. Column purification of the thus obtainedcrude product provided the pure product as light brown solid. Yield 2 g(64%). ¹H NMR (300 MHz) δ 1.03 (t, 3H), 1.80-1.92 (m, 2H), 2.65 (s, 3H),2.76 (t, 2H).

Method 596-Benzyl-3-methyl-5-propyl-6H-isothiazolo[4,5-d]pyrimidin-7-one

3-Methyl-5-propyl-isothiazolo[4,5-d][1,3]oxazin-7-one (method 58) (200mg, 1.02 mmol) was taken in a 10 mL microwavable pyrex tube and benzylamine (1 g, 9.34 mmol) was added to it. The resulting mixture was heatedin a microwave synthesizer (CEM's Discoverer) at 200° C. for 20 min. TheMS of the reaction mixture showed the complete disappearance of thestarting material and the presence of the product peak at 286 (MH⁺). Thereaction mixture was diluted with 1N HCl (10 mL) and extracted withEtOAc (2×30 mL). The combined EtOAc layers were washed with water,brine, dried and concentrated. The thus obtained crude product waspurified by column chromatography to isolate the pure product as a whitesolid. Yield 208 mg (71%). ¹H NMR (300 MHz) δ 0.98 (t, 3H), 1.76-1.88(m, 2H), 2.68 (s, 3H), 2.74 (t, 2H), 5.42 (s, 2H), 7.10-7.19 (m, 2H),7.28-7.39 (m, 3H).

Method 606-Benzyl-5-(1-bromo-propyl)-3-methyl-6H-isothiazolo[4,5-d]pyrimidin-7-one

To a solution of6-benzyl-3-methyl-5-propyl-6H-isothiazolo[4,5-d]pyrimidin-7-one (method59) (208 mg, 0.69 mmol) and sodium acetate (0.5 g, 5 mmol) in aceticacid (10 mL) at 100° C., a solution of the bromine (0.232 g, 1.46 mmol)in acetic acid (20 mL) was added dropwise [The next drop of Bromine wasadded only after the previous drop had reacted completely by monitoringthe decolorization] over a period of 30 min. The reaction mixture wascooled after the addition and the TLC (eluent 10% EtOAc in hexanes) andMS showed the complete disappearance of the SM and only the product. Thereaction mixture was poured into ice water and extracted with EtOAc(3×30 mL) and the organic layers were combined and washed with 2% sodiumthiosulfate solution (30 mL), water (50 mL), brine (50 mL) and dried(Na₂SO₄). Concentration of the organic layer provided the product and itwas pure enough to be used in the next step. Yield 260 mg (99%). ¹H NMR(300 MHz) δ 0.77 (t, 3H), 2.20-2.54 (m, 2H), 2.70 (s, 3H), 4.67 (t, 1H),4.95 (d, 1H), 6.25 (d, 1H) 7.10-7.19 (m, 2H), 7.30-7.39 (m, 3H).

Method 61N-(3-Amino-propyl)-N-[1-(6-benzyl-3-methyl-7-oxo-6,7-dihydro-isothiazolo[4,5-d]pyrimidin-5-yl)-propyl]-4-methyl-benzamideHydrogen Chloride

To a solution of6-benzyl-5-(1-bromo-propyl)-3-methyl-6H-isothiazolo[4,5-d]pyrimidin-7-one(method 60) (260 mg, 0.70 mmol) in anhydrous DMF (10 mL), ethyldiisopropylamine (387 mg, 3 mmol) and N-(3-aminopropyl)carbamic acidtert-butyl ester (174 mg, 1 mmol) were added at room temperature and themixture was stirred at room temperature for 1 h after which the MSanalysis showed the complete disappearance of the starting bromide andonly the product peak at 472 (MH⁺) was observed. The reaction mixturewas diluted with water (100 mL) and extracted with EtOAc (3×60 mL). Thecombined organic extracts were dried and concentrated to get the crudeamine which was dissolved in chloroform (40 mL) anddiisopropylethylamine (387 mg, 3 mmol) was added and the mixture washeated to 60° C. To the stirred hot solution p-toluoyl chloride (154 mg,1 mmol) in chloroform (20 mL) was added dropwise and the mixture wasrefluxed for 12 h after which the MS showed the complete disappearanceof the amine and only the product peak at 590 (MH⁺). The reactionmixture was concentrated and the crude product was purified by columnchromatography to isolate the pure acylated product (80 mg, 20% overallfrom bromide) which was treated with 4M HCl in 1,4-dioxane (10 mL) for30 min. The dioxane was evaporated in a rotary evaporator and theresidue was dissolved in water and freeze dried to get the pure productas a white fluffy solid. Yield 60 mg (16% overall from bromide). m/z 490(MH⁺); ¹HNMR (300 MHz, DMSO-d₆, 96° C.) δ 0.65 (t, 3H), 1.36-1.50 (m,1H), 1.60-1.72 (m, 1H), 1.88-1.99 (m, 1H), 2.14-2.26 (m, 1H), 2.35 (s,3H), 2.47 (t, 2H), 2.68 (s, 3H), 3.32-3.44 (m, 2H), 4.90 (d, 1H), 5.50(bs, 1H), 5.76 (d, 1H), 6.96-7.34 (m, 9H), 7.68 (bs, 3H).

Method 62 Chiral Purification of (+)N-(3-Amino-propyl)-N-[1-(6-benzyl-3-methyl-7-oxo-6,7-dihydro-isothiazolo[4,5-d]pyrimidin-5-yl)-propyl]-4-methyl-benzamide

The following compound was chirally purified in same manner as (+)(3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-(4-methyl-benzoyl)-amino]-propyl)-carbamicacid tert-butyl ester (method 12). Chiral purification generallyresulted in 99% purity of the (+) enantiomer.

Method Column Solvent (+) Enantiomer # Compound Name Type compositionretention time SM 62 (+) N-(3-Amino- Chiralpak 70% hexane 11.7 minMethod propyl)-N-[1-(6-benzyl- AD 30% 61 3-methyl-7-oxo-6,7- isopropanoldihydro-isothiazolo[4,5- 0.1% d]pyrimidin-5-yl)- diethylaminepropyl]-4-methyl- benzamide Example G-1

Alternative Procedures to Prepare Certain Starting Materials Method 12-(1-Ethoxy-ethylidene)-malononitrile (Alternative Procedure)

Triethyl orthoacetate (1.6 L, 9 mol), malononitrile (500 g, 7.57 mol)and glacial acetic acid (25 ml) were placed in a 5 l RB flask equippedwith a stirrer, thermometer and a Vigreux column (20×1 in.) on top ofwhich a distillation condenser was placed. The reaction mixture washeated and ethyl alcohol began to distil when the temperature of thereaction mixture was about 85-90° C. After about 3 h., the temperatureof the reaction mixture reached 140° C. Then the reaction wasconcentrated in a rotary evaporator to remove the low-boiling materialsand the residue was stirred with isopropyl alcohol (1 l) and cooled inan ice bath. The crystallized product was filtered off washed withisopropyl alcohol (200 ml), hexanes (600 ml) and dried at 50° C. in avacuum oven overnight to yield 2-(1-ethoxy-ethylidene)-malononitrile(974 g, 94%) as a golden yellow solid [mp 92. ° C. (lit. 90-92° C.,MCCall. M. A. J. Org. Chem. 1962, 27, 2433-2439.)].

Method 2 (2E)-2-Cyano-3-ethoxybut-2-enethioamide (Alternative Procedure)

2-(1-Ethoxy-ethylidene)-malononitrile (method 1) (300 g, 2.2 mol) wasdissolved in anhydrous benzene (3.11, slight warming required) and 20 mlof triethylamine was added. The mixture was mechanically stirred andhydrogen sulfide was bubbled into this solution for 2 h and a solidformed. Then N₂ was bubbled through the reaction mixture for 40 min. Theprecipitated solid was filtered off, washed with cold benzene (200 ml)and dried in a vacuum oven overnight to isolate(2E)-2-cyano-3-ethoxybut-2-enethioamide (332 g, 88%) as light browncrystals.

Method 3 (2E)-3-Amino-2-cyanobut-2-enethioamide (Alternative Procedure)

(2E)-2-Cyano-3-ethoxybut-2-enethioamide (method 2) (150 g, 0.88 mol) wasdissolved in 7M solution of ammonia in methanol (2.9 L) and stirred atr.t. overnight. The reaction mixture was concentrated and the residuewas crystallized from hot water (1. L) to provide(2E)-3-amino-2-cyanobut-2-enethioamide (111.6 g, 89%) as brown crystals.¹H NMR (300 MHz, DMSO-d₆) δ 2.22 (s, 3H), 7.73 (bs, 1H), 8.53 (bs, 1H),9.01 (bs, 1H), 11.60 (bs, 1H).

Method 4 5-Amino-3-methylisothiazole-4-carbonitrile (AlternativeProcedure)

To a stirred solution of (2E)-3-amino-2-cyanobut-2-enethioamide (method3) (111 g, 0.78 mol) in methanol (2 L) was added dropwise 200 ml of 35%hydrogen peroxide over a period of 30 min. After the completion of theaddition the mixture was stirred at 60° C. for 3 h after which the TLCshowed the completion of the reaction. The reaction mixture wasevaporated to 300 ml in a rotary evaporator and cooled in an ice-bath.The crystallized product was filtered off and washed with isopropylalcohol (100 ml) and dried in vacuum at 50° C. overnight to provide5-amino-3-methylisothiazole-4-carbonitrile (105.63 g, 96%) as a lightyellow crystalline solid. ¹H NMR (300 MHz, DMSO-d₆) δ 2.24 (s, 3H), 8.00(bs, 2H).

Method 24 N-(4-Cyano-3-methyl-isothiazol-5-yl)-3-methyl-butyramide(Alternative Procedure)

To a solution of 5-amino-3-methylisothiazole-4-carbonitrile (method 4)(105.6 g, 0.76 mol) in pyridine (250 ml) at 0° C., isovaleryl chloride(100 g, 0.83 mol) in chloroform (300 ml) was added dropwise. After thecompletion of the addition the reaction mixture was allowed to warm tor.t. and stirred overnight. The TLC and the MS showed the completedisappearance of the starting material and the reaction mixture wasdiluted with CHCl₃ (600 ml), washed with water (200 ml), 2N HCl (600ml), satd. NaHCO₃ (200 ml), brine (200 ml) and dried over Na₂SO₄.Concentration of the CHCl₃ layer provided the crude product which wastriturated from DCM/hexanes ( 1/10) and filtered off to isolateN-(4-cyano-3-methyl-isothiazol-5-yl)-3-methyl-butyramide (149.7 g, 88%)as an off-white crystalline solid. ¹H NMR (300 MHz) δ 1.04 (d, 6H),2.18-2.32 (m, 1H), 2.46 (d, 2H), 2.53 (s, 3H), 9.87 (bs, 1H).

Method 25 3-Methyl-5-(3-methyl-butyrylamino)-isothiazole-4-carboxylicacid amide (Alternative Procedure)

To a solution ofN-(4-cyano-3-methyl-isothiazol-5-yl)-3-methyl-butyramide (method 24) (72g, 322 mmol) in 30% aqueous NH₄OH (2.1 L), was added dropwise 1.3 L ofhydrogen peroxide at 40° C. After 20 min the temperature of the reactionmixture rose to 60° C. The addition was completed in 1.5 h. After anadditional 2 h the MS showed the completion of the reaction. Thereaction mixture was cooled in ice and con HCl was slowly added withcooling till the pH of the reaction mixture turns 7.6. The precipitatedproduct was filtered and dried in vacuum oven to get the pore amide (36g, 46%). The filtrate was saturated with NaCl and extracted with supersolvent (34:66, t-butanol: 1,2-dichloroethane) and the combined organicextracts were washed with water (500 ml), brine (600 ml) and dried(Na₂SO₄) and concentrated. The residue on trituration with EtOAc/hexanes(1/4) provided an additional 9.8 g of pure product. Total yield of 45.8g (58%) 3-methyl-5-(3-methyl-butyrylamino)-isothiazole-4-carboxylic acidamide. ¹H NMR (300 MHz) δ 1.03 (d, 6H), 2.24 (m, 1H), 2.43 (d, 2H), 2.69(s, 3H), 5.98 (bs, 2H), 11.77 (bs, 1H).

Method 26 6-Isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(Alternative Procedure)

The 3-methyl-5-(3-methyl-butyrylamino)-isothiazole-4-carboxylic acidamide (method 25) (45.8 g, 190 mmol) was suspended in 700 ml of 30% NH₃and then was heated to 140° C. for 5 h in a pressure reactor. Themixture was poured into a 4 L beaker and cooled in an ice bath. To thecold solution con HCl (560 ml) was added dropwise to pH 7.5 and a whiteprecipitate was formed. The precipitated product was filtered off,washed with water (100 ml) and dried under vacuum overnight.6-Isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one (11 g, 26%) wasisolated as an off-white powder. ¹H NMR (300 MHz) δ 1.05 (d, 6H), 2.32(m, 1H), 2.69 (d, 2H), 2.82 (s, 3H).

Method 275-Benzyl-6-isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(Alternative Procedure)

To a solution of the6-isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one (method 26) (11g, 49 mmol) in 60 ml of anhydrous DMF at 0° C., was added 13.8 g (100mmol) of anhydrous K₂CO₃ followed by benzyl bromide (9.3 g, 54 mmol) andthe mixture was stirred at 0-20° C. overnight. The TLC of the reactionmixture showed the complete disappearance of the SM. The reactionmixture was poured into ice-cold water and extracted with EtOAc (3×100ml). The combined extracts were washed with water (100 ml), brine (100ml), dried (Na₂SO₄) and concentrated. The TLC and the ¹H NMR showed thepresence of two products N alkylated as well as O-alkylated products ina ratio of 75:25. The products were separated by column (silica gel)chromatography using 10% EtOAc in hexanes. The major N-alkylated product5-benzyl-6-isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one wasisolated as white crystalline solid (10.8 g, 70%). ¹H NMR (300 MHz) δ0.94 (d, 6H), 2.23-2.37 (m, 1H), 2.64 (d, 2H), 2.82 (s, 3H), 5.38 (s,2H), 7.10-7.38 (m, 5H).

Method 285-Benzyl-6-(1-bromo-2-methyl-propyl)-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(Alternative Procedure)

To a solution of5-benzyl-6-isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 27) (5.81 g, 18.5 mmol) and sodium acetate (10 g) in acetic acid(100 ml) at 100° C., a solution of the bromine (6 g, 38 mmol) in aceticacid (60 ml) was added dropwise over a period of 20 minutes. Thereaction mixture was stirred at that temperature for 30 min and cooledand the TLC (eluent 10% EtOAc in hexanes) and MS showed the completedisappearance of the SM and only the product. The reaction mixture waspoured into ice water and extracted with EtOAc (3×60 ml) and the organiclayers were combined and washed with 2% sodium thiosulfate solution (60ml), water (100 ml), brine (100 ml) and dried over Na₂SO₄. Concentrationof the organic layer provided5-benzyl-6-(1-bromo-2-methyl-propyl)-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(7.27 g, 99%) as white crystalline solid. ¹H NMR (300 MHz) δ 0.54 (d,3H), 1.11 (d, 3H), 2.62-2.76 (m, 1H), 2.83 (s, 3H), 4.42 (d, 1H), 4.80(d, 1H), 6.22 (d, 1H), 7.12-7.42 (m, 5H).

Method 296-(1-Azido-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(Alternative Procedure)

To a solution of5-benzyl-6-(1-bromo-2-methyl-propyl)-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 28) (7.27 g, 18.5 mmol) in anhydrous DMF (60 ml), sodium azide(2.33 g, 37 mmol) was added and the mixture was stirred at roomtemperature for 2 hour. The TLC of the RM showed the completedisappearance of the starting bromide. The reaction mixture was pouredinto ice water (300 ml) and extracted with EtOAc (3×100 ml). The organiclayer was washed with water (100 ml), brine (100 ml) and dried (Na₂SO₄).Concentration of the organic layer provided the crude product which waspurified by column (silica gel) chromatography using 30% EtOAc inhexanes as eluent to isolate6-(1-azido-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(6.16 g, 94%) as a low melting solid. ¹H NMR (300 MHz) δ 0.57 (d, 3H),1.07 (d, 3H), 2.50-2.74 (m, 1H), 2.98 (s, 3H), 3.71 (d, 1H), 5.05 (d,1H), 5.78 (d, 1H), 7.12-7.40 (m, 5H).

Method 306-(1-Amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(Alternative Procedure)

To a solution of6-(1-azido-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 29) (6.8 g, 19.2 mmol) in methanol (400 ml) was added 5% Pd/C (1g, 20% by wt.) and the resulting mixture was stirred at r.t. in anatmosphere of H₂ and the progress of the reaction was monitored by MS.After the disappearance of the starting material the reaction mixturewas filtered through celite and washed with EtOAc. Concentration of thefiltrate provided6-(1-amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(5.42 g, 86%).

Method 31{3-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propylamino]-propyl}-carbamicAcid Tert-Butyl Ester (Alternative Procedure)

To a solution of6-(1-amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 30) (5.4 g, 16.5 mmol) in DCM (100 ml), 4 Å molecular sieves (50g) was added followed by N-boc protected 3-aminopropanal (2.84 g, 16.5mmol)) and the reaction mixture was stirred at r.t. overnight and theprogress of the reaction was monitored by MS. After the completedisappearance of the starting amine, a catalytic amount of acetic acidwas added to the reaction followed by sodium triacetoxyborohydride (3.49g, 16.5 mmol) and the reaction mixture was stirred at r.t. for 4 h.After the completion of the reaction (MS), the reaction mixture wasfiltered and the residue was washed with DCM and the filtrate was washedwith water (100 mL), brine (100 mL) and concentrated to give{3-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propylamino]-propyl}-carbamicacid tert-butyl ester (8.3 g, theoretical yield=7.9 g) which was used assuch for the next reaction.

Method 33{3-[[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicAcid Tert-Butyl Ester (Alternative Procedure)

To a solution of{3-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propylamino]-propyl}-carbamicacid tert-butyl ester obtained from method 31 alternative procedureabove in chloroform (300 ml), diisopropylethylamine (6 g, 46.5 mmol) wasadded and the reaction mixture was heated to 60° C. To the hot solutiona solution of the p-toluoyl chloride (3.78 g, 24.4 mmol) in chloroform(150 ml) was added dropwise and the resulting solution was refluxedovernight. The TLC showed the disappearance of most of the SM. Thereaction mixture was washed with water (2×100 ml), satd, NaHCO₃ (200 ml)brine (100 ml) and dried (Na₂SO₄). Concentration of the organic layerprovided the crude product which was purified by column (silica gel)chromatography using 10-30% EtOAc in hexanes as eluent. Yield=6.14 g(62%) of{3-[[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicacid tert-butyl ester. White foam, mp. 70-71° C. m/z 604 (MH⁺), ¹H NMR(DMSO-d₆, 300 MHz, 95° C.) δ: 0.48 (d, 3H), 0.90 (d, 3H), 1.26 m, 1H),1.28 (s, 9H), 2.33 (s, 3H), 2.47 (d, 2H), 2.72-2.64 (m, 1H), 2.72 (s,3H), 3.24 (t, 2H), 5.08 (d, 1H), 5.60 (d, 1H), 5.90 (d, 1H), 7.20-7.40(m, 9H).

Method 63 5-Amino-3-methylisothiazole-4-carboxamide

To a chilled solution of sulfuric acid (7.2 volumes, 12.9 equivs) wascharged 5-amino-3-methylisothiazole-4-carbonitrile (method 4) (1.0equiv). The temperature was maintained below 55° C. The reaction mixturewas heated to 70° C. and held for 1 hour until TLC showed disappearanceof starting material. The mixture was cooled to 60-65° C. before theammonia (21 volumes) was charged to pH 10. The mixture was cooled to 20°C., aged overnight and filtered. The resulting solid was washed withdilute ammonia (3.6 volumes) and dried at 40° C. to give a pale brownsolid (typical yield 80%). ¹H NMR (300 MHz, DMSO-d₆) δ 2.46 (s, 3H),6.28 (s, 1H).

Method 26 6-Isobutyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(Alternative Procedure)

To a 2 L flask equipped with Dean Stark was charged5-amino-3-methylisothiazole-4-carboxamide (method 63) (1 equiv),p-toluene sulphonic acid (0.049 equiv), DMF (9.75 volumes). The reactionwas stirred until a solution was obtained and isovaleraldehyde (1.10equiv) and toluene (4.9 volumes) were added. The resulting mixture washeated to 130° C. and held at reflux for 1 hour removing water via aDean Stark apparatus. Once the reaction was complete toluene was removedunder vacuum distillation. Sodium bisulfite (2.50 equiv) was charged andthe mixture was held at 115° C. for 7 hours, then cooled to roomtemperature overnight. The solid was removed by filtration throughharborlite and washed with DMF (1 volume). Analysis showed conversion toproduct and the reaction was heated to 5° C., water (15 volumes) wasadded and the resulting precipitate was cooled to room temperature andheld for 1 h. The product was isolated by filtration and washed withwater (2×0.5 volumes), dried to give a pale brown solid (typical yield89%).

Method 31{3-[1-(5-Benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-2-methyl-propylamino]-propyl}-carbamicAcid Tert-Butyl Ester (Alternative Procedure)

To (3,3-diethoxypropyl)amine (1.00 equiv) in THF (2 volumes) was chargeddi-t-butyldicarbonate (1.05 equiv) in THF (3 volumes). The reaction washeated to 45° C. and held for ½ h. Analysis showed the disappearance ofstarting material, and the resulting solution was heated to 65° C.p-Toluene sulphonic acid (0.1 equiv) and water (5 volumes) were chargedover 10 mins, heating continued at 65° C. and held for ½ hour. Analysisshowed disappearance of tert-butyl (3,3-diethoxypropyl)carbamate.Toluene (15 volumes) charged, layers separated and washed with water (5volumes). A fraction of the solution obtained (0.95 equivs) was chargedto a solution containing6-(1-amino-2-methyl-propyl)-5-benzyl-3-methyl-5H-isothiazolo[5,4-d]pyrimidin-4-one(method 30) (1 equiv), toluene (5 volumes) and molecular sieves (1weight equivalent). The reaction mixture was stirred overnight at roomtemperature until the reaction was complete. THF (2.5 volumes) werecharged followed by sodium acetoxyborohydride (2.0 equiv) and theresulting mixture held overnight until reaction was complete. Aqueousacetic acid (20% v/v, 2.5 volumes) were charged over 10 minutes, stirredat room temperature for 10 minutes, filtered and washed with water (2.5volumes). The layers were separated and the organic layer wasconcentrated under vacuo at 50° C. Further toluene was charged (2.5volumes) and the solvent removed. The product was obtained as an orangeoil (typical yield 92%). m/z 486 (MH⁺).

Example A

Examples A

The following compounds were synthesized according to synthetic scheme Aabove:

Ex. Compound ¹H NMR m/z SM A1 (+) N-(3-Amino-propyl)- (DMSO-d₆, 500 MHz,96° C.) δ: m/z Method N-[1-(5-benzyl-3-methyl- 0.63 (t, 3H), 1.40-1.74(m, 2H), 490 (MH⁺) 13 4-oxo-4,5-dihydro- 1.75-1.96 (m, 1H), 2.05-2.20(m, 1H), 2.39 (s, isothiazolo[5,4- 3H), 2.46 (t, 2H), 2.72 (s, 3H),d]pyrimidin-6-yl)-propyl]- 3.36 (t, 2H), 4.83 (d, 1H), 5.50 (bs, 1H),4-methyl-benzamide 5.77 (d, 1H), 6.95-7.37 (m, 9H), hydrogen chloride7.79 (bs, 3H) A2 (+) N-(3-Amino-propyl)- (DMSO-d₆, 500 MHz, 96° C.) δ:m/z Method N-{1-[5-(4-fluoro-benzyl)- 0.66 (t, 3H), 1.38-1.74 (m, 2H),508 (MH⁺) 13a 3-methyl-4-oxo-4,5- 1.82-1.98 (m, 1H), 2.02-2.20 (m, 1H),2.34 (s, dihydro-isothiazolo[5,4- 3H), 2.42 (t, 2H), 2.72 (s, 3H),d]pyrimidin-6-yl]-propyl}- 3.36 (t, 2H), 4.85 (d, 1H), 5.49 (bs, 1H),4-methyl-benzamide 5.70 (d, 1H), 7.05-7.27 (m, 8H), hydrogen chloride7.76 (bs, 3H) A3 (+) N-(3-Amino-propyl)- (500 MHz, DMSO-d₆, 100° C.) δm/z Method N-{1-[5-(3-fluoro-benzyl)- ppm: 0.70 (t, 3H), 1.40-1.54 (m,508 (MH⁺) 13b 3-methyl-4-oxo-4,5- 1H), 1.62-1.76 (m, 1H),dihydro-isothiazolo[5,4- 1.85-2.01 (m, 1H), 2.14-2.27 (m, 1H), 2.38 (s,d]pyrimidin-6-yl]-propyl}- 3H), 2.44-2.49 (m, 2H), 2.76 (s,4-methyl-benzamide 3H), 3.35-3.46 (m, 2H), 4.87 (br s, hydrogen chloride1H), 5.48 (br s, 1H), 5.75 (d, 1H), 6.84-6.96 (m, 2H), 7.06-7.15 (m,1H), 7.20-7.31 (m, 4H), 7.33-7.41 (m, 1H), 7.52 (br s, 3H) A4 (+)N-(3-Amino-propyl)- (DMSO-d₆, 500 MHz, 96° C.) δ: m/z MethodN-[1-(5-benzyl-3-methyl- 0.68 (t, 3H), 1.50-1.72 (m, 2H), 554, 13g4-oxo-4,5-dihydro- 1.91-1.96 (m, 1H), 2.13-2.17 (m, 1H), 2.47 (t, 556(MH⁺) isothiazolo[5,4- 2H), 2.77 (s, 3H), 3.38 (t, 2H),d]pyrimidin-6-yl)-propyl]- 4.95 (d, 1H), 5.57 (bs, 1H), 5.80 (d, 1H),4-bromo-benzamide 7.13 (m, 2H), 7.28-7.36 (m, 5H), hydrogen chloride7.64 (d, 2H), 7.80 (br, 1H) A5 (+) N-(3-Amino-propyl)- (DMSO-d₆, 500MHz, 96° C.) δ: m/z Method N-[1-(5-benzyl-3-methyl- 0.69 (t, 3H),1.42-1.83 (m, 2H), 510 (MH⁺) 13c 4-oxo-4,5-dihydro- 1.89-2.01 (m, 1H),2.10-2.20 (m, 1H), isothiazolo[5,4- 2.46 (hidden by DMSO, 2H), 2.77 (s,d]pyrimidin-6-yl)-propyl]- 3H), 3.39 (bm, 2H), 4.94 (d, 1H),4-chloro-benzamide 5.58 (bs, 1H), 5.81 (d, 1H), hydrogen chloride7.12-7.56 (m, 9H) A6 (+) N-(3-Amino-propyl)- (DMSO-d₆, 500 MHz, 96° C.)δ: m/z Method N-[1-(5-benzyl-3-methyl- 0.67 (t, 3H), 1.45 (m, 1H), 1.70(m, 1H), 508 (MH⁺) 13d 4-oxo-4,5-dihydro- 1.92 (m, 1H), 2.16 (m, 1H),2.31 (s, isothiazolo[5,4- 3H), 2.46 (2H, hidden by DMSO),d]pyrimidin-6-yl)-propyl]- 2.76 (s, 3H), 3.39 (t, 2H), 4.93 (d,3-fluoro-4-methyl- 1H), 5.54 (bs, 1H), 5.81 (d, 1H), benzamide hydrogen7.09-7.52 (m, 8H), 7.74 (br, 3H) chloride A7 (+) N-(3-Amino-propyl)-(DMSO-d₆, 500 MHz, 96° C.) δ: m/z Method N-[1-(5-benzyl-3-methyl- 0.70(t, 3H), 1.50-1.70 (m, 2H), 1.94 (m, 544, 13e 4-oxo-4,5-dihydro- 1H),2.20 (m, 1H), 2.46 (m, 2H), 545, isothiazolo[5,4- 2.78 (s, 3H), 3.30 (m,2H), 5.10 (d, 546 (MH⁺) d]pyrimidin-6-yl)-propyl]- 1H), 5.82 (bs, 1H),5.93 (d, 1H), 2,3-dichloro-benzamide 7.29-7.72 (m, 8H), 7.72 (br, 3H)hydrogen chloride A8 (+) Benzo[b]thiophene-2- (DMSO-d₆, 500 MHz, 96° C.)δ: m/z Method carboxylic acid (3-amino- 0.71 (t, 3H), 1.46-1.61 (m, 1H),532 (MH⁺) 13f propyl)-[1-(5-benzyl-3- 1.88-1.92 (m, 1H), 1.93-1.97 (m,1H), methyl-4-oxo-4,5-dihydro- 2.21-2.26 (m, 1H), 2.62 (t, 2H), 2.76 (s,isothiazolo[5,4- 3H), 3.65 (t, 2H), 4.96 (d, 1H), d]pyrimidin-6-yl)-5.66 (bs, 1H), 5.78 (d, 1H), 7.05 (bm, propyl]amide hydrogen 2H), 7.25(bm, 3H), 7.30-7.50 (m, chloride 2H), 7.60 (s, 1H), 7.85-7.99 (m, 2H) A9(+) N-(2-Amino-ethyl)-N- (DMSO-d₆, 500 MHz, 96° C.) δ: m/z Method[1-(5-benzyl-3-methyl-4- 0.60 (t, 3H), 1.85-2.05 (m, 2H), 2.45 (s, 476(MH⁺) 13h oxo-4,5-dihydro- 3H), 2.76 (s, 3H), 2.90 (m, 2H),isothiazolo[5,4- 3.80 (m, 2H), 4.70 (d, 1H), 5.35 (bs, 1H),d]pyrimidin-6-yl)-propyl]- 5.80 (d, 1H), 6.88-7.35 (m, 9H),4-methyl-benzamide 7.75-7.85 (br, 3H) hydrogen chloride A10 (+)N-[1-(5-Benzyl-3- (DMSO-d₆, 90° C.) δ: 0.66 (t, 3H), m/z Methodmethyl-4-oxo-4,5-dihydro- 0.90-1.10 (m, 1H), 1.30-1.49 (m, 518 (MH⁺) 12iisothiazolo[5,4- 1H), 1.81 (m, 8H), 1.85-1.95 (m,d]pyrimidin-6-yl)-propyl]- 1H), 2.05-2.15 (m, 1H), 2.35 (s, 3H),N-(3-dimethylamino- 2.75 (s, 3H), 3.41 (t, 2H), 4.96 (d,propyl)-4-methyl- 1H), 5.71 (bs, 1H), 5.92 (d, 1H), benzamide 7.10-7.44(m, 9H)

Example B

Example B

The following compounds were synthesized according to synthetic scheme Babove:

Ex. Compound ¹H NMR m/z SM B1 (+) N-[1-(5-Benzyl-3-methyl- (DMSO-d₆, 90°C.) δ: 0.65 (t, 3H), m/z Method 4-oxo-4,5-dihydro- 0.75-0.85 (d, 6H),1.01-1.11 (m, 532 (MH⁺) 15 isothiazolo[5,4-d]pyrimidin- 1H), 1.35-1.50(m, 1H), 6-yl)-propyl]-N-(3- 1.80-1.98 (m, 1H), 2.00-2.19 (m, 3H), 2.35(s, isopropylamino-propyl)-4- 3H), 2.80 (s, 3H), 3.00-3.05 (b, 2H),methyl-benzamide 3.40 (m, 2H), 4.90 (d, 1H), 5.70 (bs, 1H), 5.80 (d,1H), 7.00-7.40 (m, 9H)

Example C

Example C

The following compounds were synthesized according to synthetic scheme Cabove:

Ex. Compound ¹H NMR m/z SM C1 (+) N-(3-Amino-propyl)-N-[1- (500 MHz,100° C., DMSO-d₆) m/z Method (5-benzyl-3-methyl-4-oxo-4,5- δ: 0.68 (t,3H), 1.52 (m, 1H), 474 (MH⁺) 23 dihydro-isoxazolo[5,4- 1.72 (m, 1H),1.92 (m, 1H), d]pyrimidin-6-yl)-propyl]-4- 2.10 (m, 1H), 2.39 (s, 3H),methyl-benzamide hydrogen 2.51 (m, 2H), 2.57 (s, 3H), 3.41 (m, chloride2H), 4.85 (br, 1H), 5.50 (br, 1H), 5.77 (d, 1H), 7.07 (br, 2H),7.24-7.35 (m, 7H), 7.73 (br, 3H)

Example D

Examples D

The following compounds were synthesized according to synthetic scheme Dabove:

Ex. Compound ¹H NMR m/z SM D1 (+) N-(3-Amino-propyl)-N- (90° C.,DMSO-d₆) δ: 0.47 (d, 3H), m/z Method {1-[5-(4-fluoro-benzyl)-3- 0.92 (d,3H), 1.10-1.28 (m, 1H), 522 (MH⁺) 35a methyl-4-oxo-4,5-dihydro-1.44-1.56 (m, 1H), 2.27 (t, 2H), isothiazolo[5,4-d]pyrimidin- 2.36 (s,3H), 2.66-2.72 (m, 1H), 2.75 (s, 6-yl]-2-methyl-propyl}-4- 3H), 3.35 (t,2H), 5.04 (d, 1H), methyl-benzamide hydrogen 5.57 (d, 1H), 5.86 (d, 1H),7.12-7.43 (m, chloride 8H), 7.71-7.81 (m, 3H) D2 (+)N-(3-Amino-propyl)-N- (500 MHz, 96° C., DMSO-d₆) δ: m/z Method[1-(5-benzyl-3-methyl-4-oxo- 0.45 (d, 3H), 0.90 (d, 3H), 1.12-1.30 (m,504 (MH⁺) 36 4,5-dihydro-isothiazolo[5,4- 1H), 1.46-1.63 (m, 1H), 2.25(t, 2H), d]pyrimidin-6-yl)-2-methyl- 2.36 (s, 3H), 2.64-2.7 (m, 1H),propyl]-4-methyl-benzamide 2.68 (s, 3H), 3.34 (t, 2H), 5.06 (d, 1H),5.59 (d, 1H), 5.90 (d, 1H), 7.20-7.40 (m, 9H), 7.71 (bs, 3H) D3 (+)N-(3-Amino-propyl)-N- (500 MHz, DMSO-d₆, 90° C.) δ: m/z Method{1-[5-(3-fluoro-benzyl)-3- 0.52 (d, 3H), 0.94 (d, 3H), 1.15-1.25 (m, 522(MH⁺) 35b methyl-4-oxo-4,5-dihydro- 1H), 1.26-1.33 (m, 1H),isothiazolo[5,4-d]pyrimidin- 1.45-1.58 (m, 1H), 2.32 (m, 2H), 2.38 (s,3H), 6-yl]-2-methyl-propyl}-4- 2.78 (s, 3H), 3.32-3.40 (m, 2H),methyl-benzamide 5.11 (bd, 1H), 5.56 (bd, 1H), 5.90-5.93 (d, 1H),7.11-7.38 (m, 8H), 7.58 (b, 2H) D4 (+) N-(2-Amino-ethyl)-N-[1- (500 MHz,DMSO-d₆, 96° C.) δ: m/z Method (5-benzyl-3-methyl-4-oxo- 0.44 (d, 3H),0.90 (d, 3H), 1.09-1.12 (m, 554, 35d 4,5-dihydro-isothiazolo[5,4- 1H),2.55-2.75 (m, 2H), 2.79 (s, 3H), 556 (MH⁺) d]pyrimidin-6-yl)-2-methyl-3.62-3.75 (m, 2H), 5.05 (m, 1H), propyl]-4-bromo-benzamide 5.60 (d, 1H),5.93 (d, 1H), hydrogen chloride 7.21-7.40 (m, 9H), 7.61 (m, 4H) D5 (+)N-(2-Amino-ethyl)-N-[1- (DMSO-d₆, 500 MHz, 90° C.) δ: m/z Method(5-benzyl-3-methyl-4-oxo- 0.39 (d, 3H), 0.93 (d, 3H), 2.40 (bm, 4H), 490(MH⁺) 35c 4,5-dihydro-isothiazolo[5,4- 2.55-2.70 (m, 2H), 2.79 (s, 3H),d]pyrimidin-6-yl)-2-methyl- 3.68-3.75 (m, 2H), 5.00 (b, 1H),propyl]-4-methyl-benzamide 5.55 (b, 1H), 5.91-5.95 (d, 1H), hydrogenchloride 7.15-7.43 (m, 9H), 7.60-7.71 (bs, 2H). D6 (+)N-(2-Amino-ethyl)-N-[1- (500 MHz, DMSO-d₆, 90° C.) δ: m/z Method(5-benzyl-3-methyl-4-oxo- 0.39 (d, 3H), 0.93 (d, 3H), 2.20-2.39 (m, 508(MH⁺) 35e 4,5-dihydro-isothiazolo[5,4- 4H), 2.60-2.70 (m, 2H), 2.79 (s,3H), d]pyrimidin-6-yl)-2-methyl- 3.63-3.74 (m, 2H), 5.00 (b, 1H),propyl]-3-fluoro-4-methyl- 5.55 (b, 1H), 5.91-5.95 (d, 1H), benzamidehydrogen chloride 7.15-7.48 (m, 8H), 7.68 (bs, 2H) D7 (+)N-(3-Amino-propyl)-N- (500 MHz, DMSO-d₆, 90° C.) δ: m/z Method[1-(5-benzyl-3-methyl-4-oxo- 0.48 (d, 3H), 0.93 (d, 3H), 1.18 (m, 1H),522 (MH⁺) 35f 4,5-dihydro-isothiazolo[5,4- 1.53 (m, 1H), 2.32-2.51 (s,m, 5H), d]pyrimidin-6-yl)-2-methyl- 2.82 (s, 4H), 3.35-3.43 (m, 2H),propyl]-3-fluoro-4-methyl- 5.10 (m, 1H), 5.62 (m, 1H), 5.94 (d, 1H),benzamide hydrogen chloride 7.11-7.38 (m, 8H), 7.51 (b, 2H) D8 (+)N-(3-Amino-propyl)-N- (DMSO-d₆, 90° C.) δ: 0.48 (d, 3H), m/z Method[1-(5-benzyl-3-methyl-4-oxo- 0.93 (m, 3H), 1.10-1.20 (m, 1H), 568, 35g4,5-dihydro-isothiazolo[5,4- 1.45-1.60 (m, 1H), 2.28-2.41 (t, 2H), 570(MH⁺) d]pyrimidin-6-yl)-2-methyl- 2.63-2.79 (m, s, 4H), 3.35-3.43 (m,propyl]-4-bromo-benzamide 2H), 5.08 (m, 1H), 5.62 (m, 1H), hydrogenchloride 5.96 (d, 1H), 7.30-7.50 (m, 7H), 7.52-7.80 (br, m, 4H)

Example E

Examples E

The following compounds were synthesized according to synthetic scheme Eabove:

Ex. Compound ¹H NMR m/z SM E1 (+) N-[1-(5-Benzyl-3- (DMSO-d₆, 90° C.) δ:0.36 (d, 3H), m/z Method methyl-4-oxo-4,5- 0.73 (m, 1H), 0.96 (d, 3H),532 (MH⁺) 40a dihydro-isothiazolo[5,4- 1.26-1.27 (m, 1H), 1.65-1.87 (brm, s, 8H), d]pyrimidin-6-yl)-2- 2.37 (s, 3H), 2.72 (m, 1H), 2.87 (s,3H), methyl-propyl]-N-(3- 3.35-3.41 (m, 2H), 5.22-5.27 (d, 1H),dimethylamino-propyl)-4- 5.73-5.76 (d, 1H), 6.12-6.17 (d, 1H),methyl-benzamide 7.22-7.41 (m, 9H) E2 (+) N-[1-(5-Benzyl-3- (DMSO-d₆,90° C.) δ: 0.36 (d, 3H), m/z Method methyl-4-oxo-4,5- 0.73 (m, 1H), 0.95(d, 3H), 596, 40 dihydro-isothiazolo[5,4- 1.20-1.23 (m, 1H), 1.64-1.82(br m, s, 8H), 598 (MH⁺) d]pyrimidin-6-yl)-2- 2.69 (m, 1H), 2.87 (s,3H), 3.35-3.37 (m, methyl-propyl]-N-(3- 2H), 5.17-5.22 (d, 1H),5.71-5.75 (d, dimethylamino-propyl)-4- 1H), 6.12-6.17 (d, 1H), 7.21-7.57(m, bromo-benzamide 9H) E3 (+) N-[1-(5-Benzyl-3- (DMSO-d₆, 90° C.) δ:0.36 (d, 3H), m/z Method methyl-4-oxo-4,5- 0.73 (m, 1H), 0.94 (d, 3H),540 (MH⁺) 40b dihydro-isothiazolo[5,4- 1.20-1.23 (m, 1H), 1.65-1.83 (brm, s, 8H), d]pyrimidin-6-yl)-2- 2.30 (s, 3H), 2.69 (m, 1H), 2.87 (s,3H), methyl-propyl]-N-(3- 3.35-3.41 (t, 2H), 5.17-5.23 (d, 1H),dimethylamino-propyl)-3- 5.71-5.74 (d, 1H), 6.11-6.16 (d, 1H),fluoro-4-methyl- 6.99-7.39 (m, 8H) benzamide

Example F

Example F

The following compounds were synthesized according to synthetic scheme Fabove:

Ex. Compound ¹H NMR m/z SM F1 (+) N-(3-Amino-propyl)-N- (500 MHz, 100°C., DMSO-d₆): δ: m/z Method [1-(5-benzyl-3-methyl-4- 0.48 (d, 3H), 0.94(d, 3H), 1.20-1.45 (m, m, 488 (MH⁺) 51 oxo-4,5-dihydro- 2H), 2.15 (m,2H), 2.38 (s, 3H), isoxazolo[5,4-d]pyrimidin- 2.58 (s, 3H), 2.70 (m,1H), 3.37 (m, 2H), 6-yl)-2-methyl-propyl]-4- 5.11 (d, 1H), 5.64 (d, 1H),5.90 (d, methyl-benzamide 1H), 7.23-7.39 (m, 9H) F2 (+)N-(3-Amino-propyl)-N- (500 MHz, 100° C., DMSO-d₆) δ: m/z Method{1-[5-(4-fluoro-benzyl)-3- 0.50 (d, 3H), 0.95-1.10 (d, m, 4H), 1.55 (m,506 (MH⁺) 51a methyl-4-oxo-4,5-dihydro- 1H), 2.32 (m, 2H), 2.40 (s, 3H),isoxazolo[5,4-d]pyrimidin- 2.60 (s, 3H), 2.75 (m, 1H), 3.40 (m, 2H),6-yl]-2-methyl-propyl}-4- 5.10 (d, 1H), 5.60 (d, 1H), 5.88 (d,methyl-benzamide 1H), 7.17-7.34 (m, 8H) F3 (+) N-(3-Amino-propyl)-N-(90° C., DMSO-d₆) δ: 0.44 (d, 3H), m/z Method {1-[5-(3-fluoro-benzyl)-3-0.96 (d, 3H), 1.15-1.35 (m, 1H), 506 (MH⁺) 50b methyl-4-oxo-4,5-dihydro-1.50-1.71 (m, 1H), 2.36 (m, s, 4H), 2.60 (s, isoxazolo[5,4-d]pyrimidin-3H), 2.60-2.80 (m, 2H), 3.43-3.54 (m, 6-yl]-2-methyl-propyl}-4- 2H),5.10 (m, 1H), 5.62 (d, 1H), methyl-benzamide 5.82 (d, 1H), 7.12-7.37 (m,8H), 7.60 (br, hydrogen chloride 3H)

Example G

Example G

The following compounds were synthesized according to synthetic scheme Gabove:

Ex. Compound ¹H NMR m/z SM G1 (+)N-(3-Amino-propyl)-N-[1- (DMSO-d₆, 90°C.) δ 0.65 (t, m/z Method (6-benzyl-3-methyl-7-oxo-6,7- 3H), 1.36-1.50(m, 1H), 490 (MH⁺) 62 dihydro-isothiazolo[4,5- 1.60-1.72 (m, 1H),1.88-1.99 (m, 1H), d]pyrimidin-5-yl)-propyl]-4- 2.14-2.26 (m, 1H), 2.35(s, 3H), methyl-benzamide 2.47 (t, 2H), 2.68 (s, 3H), 3.32-3.44 (m, 2H),4.90 (d, 1H), 5.50 (b, 1H), 5.76 (d, 1H), 6.96-7.34 (m, 9H), 7.68 (bs,3H).

Chiral Rotations of the Examples

Rotations were measured on a Perkin Elmer Model 341 polarimeter. Thecompounds were dissolved to a concentration of 1 mg/ml in methanol andthe measurements were made at 20.0° C., at 589 nM. 1 ml of solution wasused.

Example Rotation A1 + C1 + D2 + A8 + D5 + A5 + A4 + D4 + D6 + A6 + A7 +F2 + F1 + A3 + A10 + A2 + A9 + D7 + D8 + D3 + D1 + B1 + E2 + E1 + E3 +G1 + F3 +

Utility

Compounds of formula (I) have been shown to inhibit the microtubulemotor protein HsEg5 in vitro. Inhibitors of Eg5 have been shown toinhibit the formation of a mitotic spindle and therefore for celldivision. Inhibitors of Eg5 have been shown to block cells in themetaphase of mitosis leading to apoptosis of effected cells, and totherefore have anti-proliferative effects. It is believed that Eg5inhibitors act as modulators of cell division and are expected to beactive against neoplastic disease such as carcinomas of the brain,breast, ovary, lung, colon, prostate or other tissues, as well asmultiple myeloma leukemias, for example myeloid leukemia, acutelymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocyticleukemia, and lymphomas for example Hodgkins disease and non-Hodgkinslymphoma, tumors of the central and peripheral nervous system, and othertumor types such as melanoma, fibrosarcoma, Ewing's sarcoma andosteosarcoma. Therefore it is believed that the compounds of formula (I)may be used for the treatment of neoplastic disease. Hence the compoundsof formula (I) and their salts and their in vivo hydrolysable esters areexpected to be active against carcinomas of the brain, breast, ovary,lung, colon, prostate or other tissues, as well as leukemias andlymphomas, tumors of the central and peripheral nervous system, andother tumor types such as melanoma, fibrosarcoma and osteosarcoma. Thecompounds of formula (I) and their salts and their in vivo hydrolysableesters are expected to be active against neoplastic disease such ascarcinomas of the brain, breast, ovary, lung, colon, prostate or othertissues, as well as multiple myeloma leukemias, for example myeloidleukemia, acute lymphoblastic leukemia, chronic myeloid leukemia,chronic lymphocytic leukemia, and lymphomas for example Hodgkins diseaseand non-Hodgkins lymphoma, tumors of the central and peripheral nervoussystem, and other tumor types such as melanoma, fibrosarcoma, Ewing'ssarcoma and osteosarcoma. It is expected that the compounds of formula(I) would most likely be used in combination with a broad range ofagents but could also be used as a single agent.

Generally, the compounds of formula (I) have been identified in theMalachite Green Assay described herein as having an IC₅₀ value of 100micromolar or less. For example compound A7 ((+)N-(3-Amino-propyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydro-isothiazolo[5,4-d]pyrimidin-6-yl)-propyl]-2,3-dichloro-benzamidehydrogen chloride) has an IC₅₀ value of 136 nM.

Compounds provided by this invention should also be useful as standardsand reagents in determining the ability of a potential pharmaceutical toinhibit Eg5. These would be provided in commercial kits comprising acompound of this invention.

Malachite Green Assay

Enzymatic activity of the Eg5 motor and effects of inhibitors wasmeasured using a malachite green assay, which measures phosphateliberated from ATP, and has been used previously to measure the activityof kinesin motors (Hackney and Jiang, 2001). Enzyme was recombinantHsEg5 motor domain (amino acids 1-369-8H is) and was added at a finalconcentration of 6 nM to 100 μl reactions. Buffer consisted of 25 mMPIPES/KOH, pH 6.8, 2 mM MgCl₂, 1 mM EGTA, 1 mM dtt, 0.01% Triton X-100and 5 μM paclitaxel. Malachite green/ammonium molybdate reagent wasprepared as follows: for 800 ml final volume, 0.27 g of Malachite Green(J. T. Baker) was dissolved in 600 ml of H₂O in a polypropylene bottle.8.4 g ammonium molybdate (Sigma) was dissolved in 200 ml 4N HCl. Thesolutions were mixed for 20 min and filtered through 0.02 μm filterdirectly into a polypropylene container. 5 μl of compound diluted in 12%DMSO was added to the wells of 96 well plates. 80 μl of enzyme dilutedin buffer solution above was added per well and incubated with compoundfor 20 min. After this pre-incubation, substrate solution containing 2mM ATP (final concentration: 300 μM) and 6.053 μM polymerized tubulin(final concentration: 908 nM) in 15 μl of buffer were then added to eachwell to start reaction. Reaction was mixed and incubated for anadditional 20 min at room temperature. The reactions were then quenchedby the addition of 150 μl malachite green/ammonium molybdate reagent,and absorbance read at 650 nanometers exactly 5 min after quench using aSpectramax Plus plate reader (Molecular Devices). Data was graphed andIC₅₀s calculated using ExCel Fit (Microsoft).

1. A method for treating cancer comprising administering to an animal inneed thereof a therapeutically effective amount of (R)N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydroisothiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide,or a pharmaceutically acceptable salt thereof, which is substantiallyfree of (S)N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydroisothiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide.2. The method of claim 1, wherein the cancer is selected from the groupconsisting of a carcinomas of the brain, breast, ovary, lung, colon andprostate, multiple myeloma leukemias, lymphomas, tumors of the centraland peripheral nervous system, melanoma, fibrosarcoma, Ewing's sarcoma,and osteosarcoma.
 3. The method of claim 1, wherein the cancer islymphoma.
 4. The method of claim 1, 2, or 3, wherein the animal is ahuman.
 5. The method of claim 1, wherein (S)N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydroisothiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamideis present in an amount of no more than 2% w/w.
 6. The method of claim1, wherein (S)N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydroisothiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamideis present in an amount of no more than 1% w/w.
 7. A method for theprophylaxis treatment of cancer, comprising administering to an animalin need thereof a therapeutically effective amount of (R)N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydroisothiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide,or a pharmaceutically acceptable salt thereof, which is substantiallyfree of (S)N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydroisothiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide.8. The method of claim 7, wherein the animal is human.
 9. The method ofclaim 7, wherein (S)N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydroisothiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamideis present in an amount of no more than 2% w/w.
 10. The method of claim7, wherein (S)N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-4,5-dihydroisothiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamideis present in an amount of no more than 1% w/w.